Genetically modified bacteria stably expressing il-10 and insulin

ABSTRACT

The current disclosure provides microorganisms, such as lactic acid bacteria (e.g.,  Lactococcus lactis ) containing an exogenous nucleic acid encoding an IL-10 polypeptide and an exogenous nucleic acid encoding a T1D-specific antigen (e.g., a proinsulin) polypeptide, wherein both exogenous nucleic acids are integrated into the bacterial chromosome. Such microbial strains are suitable for human therapy. The disclosure further provides compositions (e.g., pharmaceutical compositions) methods of using the microorganisms and compositions, e.g., for the treatment of type 1 diabetes (T1D), including those with residual beta-cell function, e.g., recent-onset T1D. The microorganism may be administered orally, delivers the microorganism into the gastrointestinal tract, where it is released and expresses the bioactive polypeptides, The methods of the present disclosure are particularly well suited for subjects possessing residual beta-cell function, e.g., for subjects with recent-onset T1D.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of the filing date of U.S.Provisional Patent Application No. 62/383,079, filed Sep. 2, 2016.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing which has been submittedelectronically in ASCII format and is hereby incorporated by referencein its entirety. Said ASCII copy, created on Aug. 30, 2017, is named205350_0032_WO_567020_ST25.txt and is 43,027 bytes in size.

BACKGROUND

Genetically modified microorganisms (e.g., bacteria) have been used todeliver therapeutic molecules to mucosal tissues. See, e.g., Steidler,L., et al., Nat. Biotechnol. 2003, 21(7): 785-789; and Robert S. andSteidler L., Microb. Cell Fact. 2014, 13 Suppl. 1: S11.

Interleukin 10 (IL-10) producing lactic acid bacteria have beenpreviously described and mucosally administered IL-10 and/or insulin orproinsulin have been described for the treatment of type-1 diabetes(T1D). See e.g., International patent application publication WO2007/063075; Robert S. et al., Diabetes 2014, 63: 2876-2887; Steidler etal., Nat. Biotechnol. 2003, 21(7): 785-789; and Takiishi T. et al., J.Clin. Invest. 2012, 122(5): 1717-1725.

However, there is still a need in the art for genetically modifiedbacterial strains that are stable, constitutively express more than onebioactive polypeptide and are suitable for clinical usage, e.g., for thetreatment of T1D. The present disclosure addresses these needs.

SUMMARY

The current disclosure provides genetically modified microorganismscontaining chromosomally integrated nucleic acids encoding IL-10 andproinsulin (PINS), methods of preparing such microorganisms, and methodsof using such microorganisms. These genetically modified microorganismscan be suitable to human therapy, including but not limited to thetreatment of diabetes, e.g., T1D.

Microorganisms and Compositions

The present disclosure provides microorganisms, e.g., Gram-positivebacteria, such as a lactic acid bacterium (LAB) containing an exogenousnucleic acid encoding an interleukin-10 (IL-10) polypeptide, and anexogenous nucleic acid encoding a T1D-specific antigen, such asproinsulin (PINS) polypeptide, wherein the exogenous nucleic acidencoding the IL-10 polypeptide and the exogenous nucleic acid encodingthe T1D-specific antigen (e.g., PINS polypeptide) are both chromosomallyintegrated, i.e., are integrated into (or located on) the bacterialchromosome.

The microorganism can be a Gram-positive bacterium, such as an LAB. TheLAB can be a Lactococcus species bacterium. In other embodiments, theLAB is a Lactobacillus species, or a Bifidobacterium species. In someembodiments, the LAB can be Lactococcus lactis. The LAB can beLactococcus lactis subspecies cremoris. Another exemplary LAB is aLactococcus lactis strain MG1363. See, e.g., Gasson, M. J., J.Bacteriol. 1983, 154: 1-9.

In some examples according to any of the above embodiments, the IL-10polypeptide is human IL-10 (hIL-10). In other examples, the IL-10 is anIL-10 variant polypeptide, e.g., including at least one point mutation,e.g., to increase expression of the IL-10 polypeptide by the bacterium.In some examples according to these embodiments the IL-10 is “mature”human IL-10 (hIL-10), i.e. without its signal peptide. In someembodiments, the hIL-10 comprises a proline (Pro) to alanine (Ala)substitution, at position 2, when counting the amino acids in the maturepeptide. See, e.g., SEQ ID NO: 1. Such polypeptides are described, e.g.,in Steidler et al., Nat. Biotechnol. 2003, 21(7): 785-789, thedisclosure of which incorporated herein by reference in its entirety. Insome examples, the IL-10 polypeptide is wild-type human IL-10. In otherexamples, the IL-10 polypeptide is wild-type human IL-10 without its ownsignal peptide and has an amino acid sequence at least 90%, at least92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% identical to SEQ ID NO: 1. In other examples, the exogenous nucleicacid encoding the IL-10 polypeptide has a nucleotide sequence at least90%, at least 92%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% identical to SEQ ID NO: 2.

In some examples according to any of the above embodiments, theT1D-specific antigen polypeptide is a PINS polypeptide, such as humanPINS (hPINS), e.g., wild-type human PINS. In some examples, the PINSpolypeptide has an amino acid sequence at least 90%, at least 92%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to SEQ ID NO: 5. The wild-type PINS may be encoded by anucleotide sequence that is at least 90%, at least 92%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical to SEQID NO: 6 or SEQ ID NO: 7. In other examples, the PINS polypeptide ishuman PINS without its signal peptide and has an amino acid sequencethat is at least 90%, at least 92%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identical to SEQ ID NO: 3. In otherexamples, the exogenous nucleic acid encoding the PINS polypeptide has anucleotide sequence at least 90%, at least 92%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:4.

In some examples according to any of the above embodiments, themicroorganism, (e.g., LAB) expresses (e.g., constitutively expresses)the IL-10 polypeptide. In other examples, the microorganism (e.g., LAB)constitutively expresses and secretes the IL-10 polypeptide (e.g.,hIL-10). In other examples according to any of the above embodiments,the LAB constitutively expresses the T1D-specific antigen polypeptide(e.g., PINS polypeptide). In other examples, the microorganism (e.g.,LAB) constitutively expresses and secretes the T1D-specific antigen(e.g., PINS) polypeptide. In yet other examples, the microorganism(e.g., LAB) constitutively expresses and secretes the IL-10 polypeptide(e.g., hIL-10) and the T1D-specific antigen (e.g., PINS) polypeptide(e.g., hPINS).

In some examples according to any of the above embodiments, theexogenous nucleic acid encoding the IL-10 polypeptide is positioned 3′of an hllA promoter (PhllA), such as a Lactococcus lactis PhllA. In someexamples according to this embodiment, the exogenous nucleic acidencoding the IL-10 polypeptide is transcriptionally regulated by thePhllA. In other examples, the LAB includes an IL-10 expression cassettecontaining a PhilA promoter (e.g., a Lactococcus lactis PhllA), an IL-10secretion sequence (e.g., positioned 3′ of the PhllA), and the exogenousnucleic acid encoding the IL-10 polypeptide (e.g., positioned 3′ of theIL-10 secretion sequence). In some examples, the IL-10 expressioncassette is chromosomally integrated. In some examples, the IL-10expression cassette is chromosomally integrated thereby replacing orpartially replacing another gene. In some examples according to thisembodiment, the IL-10 expression cassette is chromosomally integrated atthe thyA locus, e.g., replacing an endogenous thyA gene, e.g., asdescribed in Steidler et al., Nat. Biotechnol. 2003, 21(7): 785-789. Insome examples according to any of the above embodiments, the IL-10secretion sequence is a nucleotide sequence encoding a secretion leaderof unidentified secreted 45-kDa protein (Usp45). Such secretion sequenceis referred to herein as SSusp45. In some examples, SSusp45 has an aminoacid sequence that is at least 90%, at least 92%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:10. In other examples, SSusp45 is encoded by a nucleic acid sequencethat is at least 90%, at least 92%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identical to SEQ ID NO: 11 or SEQ IDNO: 12. In some examples SSusp45 in the IL-10 expression cassette isencoded by a nucleic acid sequence that is at least 90%, at least 92%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to SEQ ID NO: 11. In some examples, the IL-10 expressioncassette is illustrated by: PhllA>>SSusp45>>hIL-10. The nucleotidesequence of an exemplary IL-10 expression cassette is depicted in FIG.11.

In some examples according to any of the above embodiments, theexogenous nucleic acid encoding the T1D-specific antigen (e.g., PINS)polypeptide is positioned 3′ of a gapB promoter, e.g., a gapB promoterthat is endogenous to the microorganism (e.g., LAB). In other examplesaccording to this embodiment, the exogenous nucleic acid encoding theT1D-specific antigen (e.g., PINS) polypeptide is transcriptionallyregulated by the gapB promoter. In other examples, the exogenous nucleicacid encoding the T1D-specific antigen (e.g., PINS) polypeptide ispositioned 3′ of a gapB gene (gapB) and its gapB promoter. In otherexamples, the LAB comprises a first polycistronic expression cassette(e.g., a dual cistron) comprising a gapB promoter positioned 5′ of agapB gene, a T1D-specific antigen secretion sequence (e.g., PINSsecretion sequence) (e.g., positioned 3′ of the gapB), and the exogenousnucleic acid encoding the T1D-specific antigen (e.g., PINS) polypeptide(e.g., 3′ of the PINS secretion sequence). In some examples according tothis embodiment, the polycistronic expression cassette further includesan intergenic region, e.g., between the gapB and the PINS secretionsequence. In some examples according to any of the above embodiments,the gapB promoter and the gapB gene are endogenous to the microorganism(e.g., LAB). In some examples, the PINS secretion sequence is SSusp45.In other examples according to the above embodiments, the firstpolycistronic expression cassette is chromosomally integrated. In otherexamples, the first polycistronic expression cassette is illustrated by:PgapB>>gapB>>intergenic region>>SSusp45>>PINS. In some examplesaccording to any of the above embodiments, the intergenic region in thefirst polycistronic expression cassette is rpmD gene 5′ intergenicregion (i.e. the region preceding rpmD). In some examples according toany of the above embodiments, the rpmD has a nucleotide sequenceaccording to SEQ ID NO: 8 (which includes a stop codon of the firstgene, and a start codon of a second gene). Without the start and stopcodons, the intergenic region rpmD has a nucleic acid sequence accordingto SEQ ID NO: 9.

In some examples according to any of the above embodiments, themicroorganism (e.g., LAB) further comprises an exogenous nucleic acidencoding a trehalose-6-phosphate phosphatase, e.g., otsB, such asEscherichia coli otsB. In some examples according to these embodiments,the exogenous nucleic acid encoding the trehalose-6-phosphatephosphatase is chromosomally integrated. In some examples, the exogenousnucleic acid encoding the trehalose-6-phosphate phosphatase ischromosomally integrated 3′ of unidentified secreted 45-kDa protein gene(usp45). In some examples according to this embodiment, the LABcomprises a second polycistronic expression cassette comprising a usp45promoter, the usp45 gene (e.g., 3′ of the promoter), and the exogenousnucleic acid encoding a trehalose-6-phosphate phosphatase (e.g., 3′ ofthe usp45 gene). In some examples, the second polycistronic expressioncassette further comprises an intergenic region between the usp45 geneand the exogenous nucleic acid encoding a trehalose-6-phosphatephosphatase. In some examples, the second polycistronic expressioncassette is illustrated by: Pusp45>>usp45>>intergenic region>>otsB. Insome examples according to these embodiments, the intergenic region isrpmD as described herein above (e.g., having SEQ ID NO: 8 or SEQ ID NO:9). The second polycistronic expression cassette may then be illustratedby: Pusp45>>usp45>>rpmD>>otsB.

In some examples according to any of the above embodiments, atrehalose-6-phosphate phosphorylase gene (trePP) is disrupted orinactivated in the microorganism (e.g., LAB). For example, the trePP hasbeen inactivated by removing the trePP gene or a fragment thereof, orthe trePP has been disrupted by inserting a stop codon. Thus, in someexamples according to these embodiments, the microorganism (e.g., LAB)lacks trePP activity.

In other examples according to any of the above embodiments, acellobiose-specific PTS system IIC component gene (ptcC) has beendisrupted or inactivated in the microorganism (e.g., LAB). For example,the ptcC has been disrupted by inserting a stop codon, or ptcC has beeninactivated by removing the ptcC or a fragment thereof. Thus, in someexamples according to these embodiments, the microorganism (e.g., LAB)lacks ptcC activity.

In other examples according to any of the above embodiments, the LABfurther comprises one or more genes encoding one or more trehalosetransporter(s). In some examples, the one or more genes encoding the oneor more trehalose transporter(s) are endogenous to the LAB. In someexamples, the LAB overexpresses the one or more genes encoding the oneor more trehalose transporter(s). In some examples according to theseembodiments, the one or more genes encoding the one or more trehalosetransporter(s) is positioned 3′ of an exogenous promoter, e.g., an hllApromoter (PhllA). For example, the one or more genes encoding the one ormore trehalose transporter(s) are transcriptionally regulated by thePhllA. In some examples according to these embodiments, the one or moregenes encoding the one or more trehalose transporter(s) is selected fromllmg_0453, llmg_0454, and any combination thereof. In some examples,llmg_0453 and llmg_0454 are transcriptionally regulated by a PhllA.

In some examples, according to any of the above embodiments, the one ormore genes encoding one or more trehalose transporter(s) comprises twogenes encoding two trehalose transporters, wherein an intergenic regionis located between the two genes. In some examples, the intergenicregion is rpmD, e.g., having SEQ ID NO: 8 or SEQ ID NO: 9. In someexamples, the microorganism (e.g., LAB) comprises a polycistronicexpression cassette comprising two nucleic acid sequences (e.g., genes)encoding two different trehalose transporters (transporter 1 andtransporter 2 sequences) and an intergenic region between the twonucleic acids encoding the two different trehalose transporters. Suchexpression cassette may be illustrated by: PhllA>>transporter1>>intergenic region>>transporter 2. In some examples according to theseembodiments, the intergenic region is rpmD as described herein above(e.g., having SEQ ID NO: 8 or SEQ ID NO: 9). The polycistronicexpression cassette may then be illustrated by:PhllA>>transporter1>>rpmD>>transporter2.

Thus, in some embodiments, the LAB comprises, in a single strain,several useful features. In one embodiment, the LAB is Lactococcuslactis, comprising:

-   (A) a chromosomally integrated promoter>>secretion    signal>>therapeutic protein, such as an interleukin, antigen, or    enzyme;-   (B) a chromosomally-integrated promoter>>secretion signal>>optional    second therapeutic protein.-   (C) a combination of mutations and insertions to promote trehalose    accumulation, which enhances LAB survivability against bile salts    and drying. The mutations are selected from    -   (i) chromosomally-integrated trehalose transporter(s), such as        PhllA>>transporter 1>>intergenic region>>transporter 2, such as        llmg_0453 and/or llmg_0454, for uptake of trehalose;    -   (ii) chromosomally-integrated Trehalose-6-phosphate phosphatase        gene (otsB; Gene ID: 1036914) positioned downstream of usp45        (Gene ID: 4797218) to facilitate conversion of        trehalose-6-phosphate to trehalose;    -   (iii) inactivated (e.g. through gene deletion)        Trehalose-6-phosphate phosphorylase gene (trePP; Gene ID:        4797140); and    -   (iv) inactivated cellobiose-specific PTS system IIC component        (Gene ID: 4796893), ptcC, (e.g. tga at codon position 30 of 446;        tga30).        The LAB may also contain an auxotrophic mutation for biological        containment, such as thyA.

In one embodiment, the LAB is Lactococcus lactis, comprising:

-   (A) a chromosomally integrated promoter>>secretion signal>>hIL-10 to    secrete mature hIL-10 from LAB, such as PhllA>>SSusp45>>hIL-10;-   (B) a chromosomally-integrated promoter>>secretion signal>>PINS, to    secrete mature PINS from LAB; such as PgapB>>gapB>>intergenic    region>>SSusp45>>PINS. The intergenic region could be, for example,    rpmD;-   (C) a combination of mutations and insertions to promote trehalose    accumulation, which enhances LAB survivability against bile salts    and drying. The mutations are selected from    -   (i) chromosomally-integrated trehalose transporter(s), such as        PhllA>>transporter 1>>intergenic region>>transporter 2, such as        llmg_0453 and/or llmg_0454, for uptake of trehalose;    -   (ii) chromosomally-integrated Trehalose-6-phosphate phosphatase        gene (otsB; Gene ID: 1036914) positioned downstream of usp45        (Gene ID: 4797218) to facilitate conversion of        trehalose-6-phosphate to trehalose;    -   (iii) inactivated (e.g. through gene deletion)        Trehalose-6-phosphate phosphorylase gene (trePP; Gene ID:        4797140); and    -   (iv) inactivated cellobiose-specific PTS system IIC component        (Gene ID: 4796893), ptcC, (e.g. tga at codon position 30 of 446;        tga30). The LAB may also contain an auxotrophic mutation for        biological containment, such as thyA.

In one embodiment, the LAB is Lactococcus lactis, with

-   (A) thyA mutation, for biological containment-   (B) a chromosomally integrated PhllA>>SSusp45>>hIL-10 to secrete    mature hIL-10 from LAB;-   (C) a chromosomally-integrated PgapB>>gapB>>intergenic region>>S    Susp45>>PINS, wherein the intergenic region is selected from rpmD,    to secrete mature PINS from LAB;-   (D) chromosomally-integrated trehalose transporter(s), such as    PhllA>>transporter 1>>intergenic region>>transporter 2, such as    llmg_0453 and/or llmg_0454, for uptake of trehalose;-   (E) inactivated (e.g. through gene deletion) Trehalose-6-phosphate    phosphorylase gene (trePP; Gene ID: 4797140);-   (F) chromosomally integrated Trehalose-6-phosphate phosphatase gene    (otsB; Gene ID: 1036914) (positioned downstream of usp45 (Gene    ID: 4797218) to facilitate conversion of trehalose-6-phosphate to    trehalose; and-   (G) inactivated cellobiose-specific PTS system IIC component (Gene    ID: 4796893), ptcC, (e.g. tga at codon position 30 of 446; tga30).

The present disclosure further provides compositions containing amicroorganism (e.g., an LAB) as described herein, e.g., a microorganism(e.g., LAB) in accordance with any of the above embodiments.

The present disclosure further provides pharmaceutical compositionscontaining a microorganism (e.g., LAB) as described herein, e.g., amicroorganism (e.g., LAB) in accordance with any of the aboveembodiments, and further containing a pharmaceutically acceptablecarrier.

The present disclosure further provides a microbial suspension (e.g.,bacterial suspension) containing a microorganism (e.g., LAB) inaccordance with any of the above embodiments, and further containing asolvent, and a stabilizing agent. In some examples according to thisembodiment, the solvent is selected from water, oil, and any combinationthereof. For example, the present disclosure provides a bacterialsuspension containing an LAB of the present disclosure, an aqueousmixture (e.g., a drink), and a stabilizing agent. Exemplary stabilizingagents are selected from a protein or polypeptide (e.g., glycoprotein),a peptide, a mono-, di- or polysaccharide, an amino acid, a gel, a fattyacid, a polyol (e.g., sorbitol, mannitol, or inositol), a salt (e.g., anamino acid salt), or any combination thereof.

The present disclosure further provides a microorganism as describedherein (e.g., an LAB in accordance with any of the above embodiments), acomposition as described herein, or a pharmaceutical composition asdescribed herein, for use in the treatment of type-1 diabetes (T1D).

The present disclosure further provides a microorganism as describedherein (e.g., an LAB in accordance with any of the above embodiments), acomposition as described herein, or a pharmaceutical composition asdescribed herein, for use in the preparation of a medicament, e.g., forthe treatment of a disease, e.g., an autoimmune disease, such as type-1diabetes (T1D)

Method 1: Methods of Treating Disease

The present disclosure further provides methods for the treatment of T1Din a subject in need thereof. Exemplary methods include administering tothe subject a therapeutically effective amount of a microorganism (e.g.,LAB) as disclosed herein (e.g., an LAB in accordance with any of theabove embodiments), a composition as disclosed herein, or apharmaceutical composition as disclosed herein. In some examplesaccording to any of these embodiments, the subject is a human, e.g., ahuman patient. In some examples according to any of these embodiments,the method further comprises administering an immunomodulatory agent(e.g., an anti-CD3 antibody) to the subject. For example, theimmuno-modulatory agent (e.g., anti-CD3 antibody) is administered to thesubject using a co-therapeutic regimen, i.e., the subject (e.g., human)is concurrently treated with another immune-modulatory agent, such as ananti-CD3 antibody. Thus, in some examples, the present disclosureprovides a method for the treatment of T1D in a human subject in needthereof. Exemplary methods include administering to the human subject atherapeutically effective amount of an LAB as disclosed herein (e.g., anLAB in accordance with any of the above embodiments), a composition asdisclosed herein, or a pharmaceutical composition as disclosed herein,wherein the human subject is further administered an anti-CD3 antibody(e.g., is concurrently treated with an anti-CD3 antibody) or fragmentthereof.

In some examples, the anti-CD3 antibody is a monoclonal antibody. Inother examples, the anti-CD3 antibody is a humanized monoclonalantibody. In other examples, the anti-CD3 antibody is otelixizumab orteplizumab. In some examples, when administered as a combination therapywith LAB, the anti-CD3 antibody is administered to the subject in a dosedifferent from that normally used for monotherapy. The optimal dose ofanti-CD3 for use in combination therapy may be determined through animalmodels and clinical trials. Preferably, the dose of anti-CD3 is lessthan a dose normally required for effective mono-therapy with anti-CD3antibody, i.e. a subtherapeutic dose. A dose effective for monotherapymay be determined by reference to animal models and clinical trials, andby the practice of those of skill in the art, and may be theregulatory-approved dose. In some examples, the subject undergoingcombination therapy with LAB is administered a dose of anti-CD3 that isat least about 10%, at least about 20%, at least about 30%, at leastabout 40%, at least about 50%, at least about 60%, at least about 70%,at least about 80%, or at least about 90% less than the standard dose ofanti-CD3 in monotherapy. In other examples, the dose of anti-CD3administered in combination therapy is about 10-100% less, about 10-90%less, about 10-70% less, about 10-60% less, about 10-50% less, about50-100% less, or about 20-50% less than the dose of anti-CD3 inmonotherapy. Experiments have shown that 6 daily doses of 48 or 64 mg ofotelixizumab can suppress T-cell activity enough that, at 36 months, thetreatment group retained 80% more beta cell function than placebocontrol. However, such a dose was associated with reactivation of latentEpstein-Barr infection. A 3.1 mg/day dose for 8 days did not preservebeta-cell function. Therefore, a treatment at least 10% less than 48 mg(i.e. 43 mg) would be considered “subtherapeutic.”

In some embodiments, the mammalian subject in the above methods, hasrecently been diagnosed with T1D (i.e., has recent-onset T1D). In someexamples according to these embodiments, the subject has been diagnosedwith T1D within the previous 12 months, the previous 24 months, or theprevious 36 months prior to administering the microorganism (e.g., LAB).

In some examples according to any of the embodiments of Method 1, themethod further includes measuring a clinical marker (e.g., an immunebiomarker) in the subject, e.g., the subject's organ or blood. Forexample, the method may further include measuring a T1D-related antibodyin the subject's blood. In some examples, the method can further includemeasuring insulin autoantibody (IAA) in the subject's blood. In someexamples, the amount of IAA in the subject's blood is indicative of T1Ddisease progression, e.g., whether the subject can be classified ashaving recent-onset T1D, and may be indicative of whether the subjectwill likely benefit from treatment. Thus, measuring IAA may occur priorto administering the microorganism to the subject. In some examples, IAApositivity at disease onset is predictive of positive therapeuticoutcome. However, measuring IAA may also be used to monitor and measurethe outcome of the treatment, and may therefore be used during treatment(e.g., throughout the treatment period in intervals) to monitortreatment efficacy or outcome and/or subsequent to the treatment period,e.g., to monitor whether the subject maintains treatment results (e.g.,normoglycemia) after treatment has stopped. In some examples, decline ofIAA positivity is indicative of a positive therapeutic outcome. In otherexamples, the method further includes measuring regulatory T cells inthe subject. Regulatory T cells include Treg17, Tr1 Th3, CD8⁺CD28⁻, Qa-1restricted T cells, CD4⁺Foxp3⁺CD25⁺ and/or CD4⁺Foxp3⁺CD25⁻ T cells.Preferably, CD4⁺Foxp3⁺CD25⁺ and/or CD4⁺Foxp3⁺CD25⁻ T cells. In otherexamples, the method further includes measuring an initial blood glucoseconcentration in the subject. In other examples, the method furtherincludes measuring C-peptide levels in the subject.

In related embodiments, the invention is a method of increasing theactivity of Treg cells to suppress the autoimmune response to beta cellsand/or a T1D-associated antigen, preferably suppressing the autoimmuneresponse to all T1D-associated antigens. In other embodiments, theinvention is a method of substantially decreasing, preferably halting,the autoimmune response to beta cells and/or any T1D-associated antigen.Preferably, the substantial decrease in the autoimmune response leads toa substantial decrease or halting of the disease progress.

In some embodiments in any of the above methods, the microorganism(e.g., LAB) is administered to the subject orally. For example, themicroorganism (e.g., LAB) is administered to the subject in the form ofa pharmaceutical composition for oral administration (e.g., a capsule,tablet, granule, or liquid) comprising the microorganism (e.g., LAB) anda pharmaceutically acceptable carrier. In other examples, themicroorganism (e.g., LAB) is administered to the subject in the form ofa food product, or is added to a food (e.g., a drink). In otherexamples, the microorganism (e.g., LAB) is administered to the subjectin the form of a dietary supplement. In yet other examples, themicroorganism (e.g., LAB) is administered to the subject in the form ofa suppository product. In some examples, the compositions of the presentdisclosure are adapted for mucosal delivery of the polypeptides, whichare expressed by the microorganism (e.g., LAB). For example,compositions may be formulated for efficient release in thegastro-intestinal tract (e.g., gut) of the subject.

In accordance with any of the above embodiments, the present disclosurefurther provides methods for establishing tolerance to a T1D-specificantigen (e.g., PINS) polypeptide in a subject in need thereof. Exemplarymethods include administering to the subject a therapeutically effectiveamount of a microorganism (e.g., LAB) as disclosed herein (e.g., an LABin accordance with any of the above embodiments), a composition asdisclosed herein, or a pharmaceutical composition as disclosed herein.In some examples according to any of these embodiments, the subject is ahuman, e.g., a human patient.

In accordance with any of the above embodiments, the present disclosurefurther provides methods for decreasing IAA in a subject in needthereof, or for increasing the number of CD4⁺Foxp3⁺ T cells in a subjectin need thereof. Exemplary methods include administering to the subjecta therapeutically effective amount of a microorganism (e.g., LAB) asdisclosed herein (e.g., an LAB in accordance with any of the aboveembodiments), a composition as disclosed herein, or a pharmaceuticalcomposition as disclosed herein. In some examples according to any ofthese embodiments, the subject is a human, e.g., a human patient.

In some examples, administering a therapeutically effective amount of amicroorganism (e.g., LAB) of the current disclosure and an anti-CD3antibody of the current disclosure to the subject (e.g., in accordancewith any of the above methods), reduces the amount of insulin thesubject is (i.e., must be) administered to control blood glucose levelsor maintain a certain blood glucose level (e.g., as recommended by aphysician, or as generally regarded as safe). For example, administeringa therapeutically effective amount of a microorganism (e.g., LAB) of thecurrent disclosure and an anti-CD3 antibody of the current disclosure tothe subject (e.g., in accordance with any of the above methods), reducesthe amount of insulin required to control blood glucose levels ormaintain a certain blood glucose level in a subject when compared to theamount of insulin required by a corresponding subject not beingadministered the microorganism (e.g., LAB) and the anti-CD3 antibody, orwhen compared to the amount of insulin required by a correspondingsubject treated with anti-CD3 antibody alone.

In other examples, administering a therapeutically effective amount of amicroorganism (e.g., LAB) of the current disclosure and an anti-CD3antibody of the current disclosure to the subject (e.g., in accordancewith any of the above methods), preserves beta-cell function in asubject (e.g., preserves the beta-cell function measured at thebeginning of treatment) in a subject, e.g., as measured using artrecognized methods. Exemplary methods for measuring beta-cell function(e.g., using C-peptide levels) are also described herein. In someexamples, beta-cell function in the subject is preserved for at leastabout 2 months, at least about 4 months, at least about 6 months, atleast about 8 months, at least about 10 months, at least about 12months, at least about 14 months, at least about 16 months, at leastabout 18 months, at least about 20 months, at least about 22 months, orat least about 24 months after administration of the microorganism(e.g., LAB) and the anti-CD3 antibody began. In other examples,administering a therapeutically effective amount of a microorganism(e.g., LAB) of the current disclosure and an anti-CD3 antibody of thecurrent disclosure to the subject (e.g., in accordance with any of theabove methods), increases the time in which beta-cell function ispreserved in the subject, when compared to the time in which beta-cellfunction is preserved in a corresponding subjects not being administeredthe microorganism (e.g., LAB) and the anti-CD3 antibody, or whencompared to a corresponding subject treated with anti-CD3 antibodyalone.

In other examples, administering a therapeutically effective amount of amicroorganism (e.g., LAB) of the current disclosure and an anti-CD3antibody of the current disclosure to the subject (e.g., in accordancewith any of the above methods), maintains normal glycemia in a subject,e.g., as measured using art recognized methods. Blood glucose rangescorrelating with normal glycemia are described herein. For example,normal glycemia in a human subject may be correlated with twoconsecutive fasting blood glucose measurements of 126 mg/dL or less.Exemplary methods for measuring blood glucose levels are known in theart and are described herein. In some examples, normal glycemia in thesubject (undergoing treatment) is preserved for at least about 2 months,at least about 4 months, at least about 6 months, at least about 8months, at least about 10 months, at least about 12 months, at leastabout 14 months, at least about 16 months, at least about 18 months, atleast about 20 months, at least about 22 months, or at least about 24months after administration of the microorganism (e.g., LAB) and theanti-CD3 antibody began. In other examples, administering atherapeutically effective amount of a microorganism (e.g., LAB) of thecurrent disclosure and an anti-CD3 antibody of the current disclosure tothe subject (e.g., in accordance with any of the above methods),increases the time in which normal glycemia is maintained in thesubject, when compared to the time normal glycemia is maintained in acorresponding subject not being administered the microorganism (e.g.,LAB) and the anti-CD3 antibody, or when compared to the time normalglycemia is maintained in a corresponding subject treated with anti-CD3antibody alone.

In other examples, administering a therapeutically effective amount of amicroorganism (e.g., LAB) of the current disclosure and an anti-CD3antibody of the current disclosure to the subject (e.g., in accordancewith any of the above methods), maintains a normal hemoglobin Alc(HbA1c) level in a subject, e.g., as measured using art recognizedmethods. In some examples, normal HbA1c level in the subject (undergoingtreatment) is maintained for at least about 2 months, at least about 4months, at least about 6 months, at least about 8 months, at least about10 months, at least about 12 months, at least about 14 months, at leastabout 16 months, at least about 18 months, at least about 20 months, atleast about 22 months, or at least about 24 months after administrationof the microorganism (e.g., LAB) and the anti-CD3 antibody.

In an alternative embodiment, the therapeutic method of the inventionameliorates the disease, such that in treated subjects the degree andfrequency of hyperglycemia is reduced compared with untreated subjects.Such reduction may be 10, 20, 30, 40, 50, 60, 70, 80 or 90% of theuntreated subjects.

Untreated T1D develops over time and becomes progressively worse as betacells in the pancreas are destroyed. Thus, as the present method canhalt disease, it is advantageous to treat subjects as early as possiblein the disease progression. Such markers of disease progression include,without limitation, glycemic index, insulin autoantibody (IAA) levels,fragment C levels, and insulitis. Thus, in a related embodiment, thetherapeutic method of the invention also includes measuring diseaseprogression prior to therapy, and during therapy.

For example, a human subject may be categorized as having early onsetT1D when any one of the following is measured in the subject: (1) twoconsecutive fasting blood glucose tests are equal to or greater than 126mg/dL; (2) when any random blood glucose measurement is greater than 200mg/dL; (3) a hemoglobin Ale (HbA1c) test that is equal to or greaterthan 6.5 percent (HbA1c is a blood test that gives a three month averageof blood sugars); or (4) a two-hour oral glucose tolerance test with anyvalue over 200 mg/dL, or combinations thereof. Thus, normal glycemia maybe characterized by any of the following: (1) two consecutive fastingblood glucose tests are below 126 mg/dL, below 120 mg/dL, or below 100mg/dL; (2) when any random blood glucose measurement is below 200 mg/dL,below 180 mg/dL, below 160 mg/dL, or below 140 mg/dL; (3) a hemoglobinAlc (HbA1c) test that is less than 6.5 percent, or less than 6%; or (4)a two-hour oral glucose tolerance test with a value below 200 mg/dL,below 180 mg/dL, below 160 mg/dL, or below 140 mg/dL, or any combinationthereof.

Once disease progression is substantially decreased (and preferablyceased, especially with regard to autoimmunity that underlies thepathogenic process), a subject may be further treated by methods torestore beta cell function. Thus in a further embodiment, the subjectmay be treated with a beta-cell transplant, and/or with drugs to causethe proliferation of endogenous beta cells. Because the method is ableto reverse the autoimmunity that underlies T1D, the method is notlimited to early onset diabetes. Once the beta-cell destroyingautoimmunity is eliminated, beta cell function can be restored throughtransplantation, and/or by encouraging the growth and development ofendogenous beta cells. Thus, the method can be applied to subjects withchronic diabetes.

In a further embodiment, the therapeutic method of the inventionameliorates disease progression, such that the rate of worsening isthree quarters, half, one quarter or less that of the untreated disease.Such worsening may be assessed through glycemic index, insulinautoantibody (IAA) levels, fragment C levels, and insulitis, forexample.

In a further embodiment, the therapeutic method of the invention isgiven to prevent T1D, such as by administration prior to any clinicalsymptoms. Preferably, the subject is identified as having T1D riskfactors, including family history, elevated (but not diabetic) sugarlevels in blood and urine, and antibodies to T1D associated antigens,and by a progressive worsening of these markers over time.

Method 2: Method of Preparing a Genetically-Modified Organism forTreatment of T1D

The current disclosure further provides methods for preparing agenetically modified microorganism (e.g., an LAB as disclosed herein.Exemplary methods include (i) contacting a microorganism (e.g., LAB)with an exogenous nucleic acid encoding an IL-10 polypeptide; and (ii)contacting the microorganism (e.g., LAB) with an exogenous nucleic acidencoding a T1D-specific antigen (e.g., PINS) polypeptide, wherein theexogenous nucleic acid encoding the IL-10 polypeptide and the exogenousnucleic acid encoding the T1D-specific antigen (e.g., PINS) polypeptideare chromosomally integrated (i.e., integrated into the chromosome ofthe microorganism, e.g., LAB). When the nucleic acids are integratedinto the microbial (e.g., bacterial) genome, e.g. in the chromosome, thegenetically modified microorganism (e.g., LAB) is formed. Themicroorganism (e.g., LAB) subjected to the genetic modification of thecurrent method can be any microbial strain, e.g., can be a wild-typebacterial strain, or can be genetically modified prior to contacting itwith the exogenous nucleic acid encoding the IL-10 polypeptide and theexogenous nucleic acid encoding the T1D-specific antigen (e.g., PINS)polypeptide.

In some examples, the above methods employ homologous recombination tointegrate the nucleic acids into the microbial (e.g., bacterial)chromosome. Thus, in some examples in accordance with these embodiments,the exogenous nucleic acid encoding the IL-10 polypeptide and theexogenous nucleic acid encoding the T1D-specific antigen (e.g., PINS)polypeptide are chromosomally integrated using homologous recombination(e.g., employing one or more integration plasmid containing therespective nucleic acids). In some examples according to any of theseembodiments, contacting the microorganism (e.g., LAB) with an exogenousnucleic acid encoding the IL-10 polypeptide (e.g., an integrationplasmid containing the exogenous nucleic acid encoding the IL-10polypeptide) occurs prior to contacting the LAB with an exogenousnucleic acid encoding the T1D-specific antigen (e.g., PINS) polypeptide(e.g., an integration plasmid containing the exogenous nucleic acidencoding the PINS polypeptide). In other examples according to any ofthese embodiments, contacting the microorganism (e.g., LAB) with anexogenous nucleic acid encoding the IL-10 polypeptide (e.g., anintegration plasmid containing the exogenous nucleic acid encoding theIL-10 polypeptide) occurs subsequent to contacting the LAB with anexogenous nucleic acid encoding the T1D-specific antigen (e.g., PINS)polypeptide (e.g., an integration plasmid containing the exogenousnucleic acid encoding the PINS polypeptide). In yet other examplesaccording to any of these embodiments, the microorganism (e.g., LAB) iscontacted concurrently with an exogenous nucleic acid encoding the IL-10polypeptide (e.g., an integration plasmid containing the exogenousnucleic acid encoding the IL-10 polypeptide) and an exogenous nucleicacid encoding the T1D-specific antigen (e.g., PINS) polypeptide (e.g.,an integration plasmid containing an exogenous nucleic acid encoding aPINS polypeptide), or a exogenous nucleic acid encoding both hIL-10 andPINS.

In some examples according to any of these embodiments, the methodfurther includes combining a culture of the genetically modifiedmicroorganism (e.g., LAB) with at least one stabilizing agent (e.g., acryopreserving agent) to form a microbial (e.g., bacterial) mixture. Insome examples, the method further includes removing water from themicrobial (e.g., bacterial) mixture forming a dried composition. Forexample, the method can further include freeze-drying the microbial(e.g., bacterial) mixture to form a freeze-dried composition. In otherexamples, the method may further include combining the geneticallymodified microorganism (e.g., LAB) or the dried composition (e.g., thefreeze-dried composition) with a pharmaceutically acceptable carrier toform a pharmaceutical composition. The method may further includeformulating the dried composition (e.g., the freeze-dried composition)or the pharmaceutical composition into a pharmaceutical dosage form.

The current disclosure further provides a genetically modifiedmicroorganism (e.g., a genetically modified LAB) prepared by a methoddescribed herein (e.g., a method in accordance with any of the aboveembodiments of Method 2).

Method 3: Method of Preparing a Pharmaceutical Composition

The disclosure further provides methods for preparing a pharmaceuticalcomposition. Exemplary methods include contacting a culture of amicroorganism (e.g., LAB) as disclosed herein (e.g., an LAB inaccordance with any of the above embodiments) with at least onestabilizing agent (e.g., a cryopreserving agent), thereby forming amicrobial (e.g., bacterial) mixture. In some examples, the at least onestabilizing agent comprises at least one cryopreserving agent. In someexamples, the microorganism (e.g., LAB) contains an exogenous nucleicacid encoding an interleukin-10 (IL-10) polypeptide, and furthercontains an exogenous nucleic acid encoding a T1D-specific antigen(e.g., PINS) polypeptide, wherein the exogenous nucleic acid encodingthe IL-10 polypeptide and the exogenous nucleic acid encoding theT1D-specific antigen (e.g., PINS) polypeptide are both chromosomallyintegrated, i.e., are integrated into (or located on) the microbial(e.g., bacterial) chromosome.

Such methods may further include removing water from the microbial(e.g., bacterial) mixture, thereby forming a dried composition. Forexample, the methods may include freeze-drying the microbial (e.g.,bacterial) mixture thereby forming a freeze-dried composition.

In some examples according to any of the above embodiments of Method 3,the method further includes contacting the dried composition (e.g., thefreeze-dried composition) with a pharmaceutically acceptable carrierforming a pharmaceutical composition. The methods may further includeformulating the dried composition (e.g., freeze-dried composition) intoa pharmaceutical dosage form, such as a tablet, a capsule, or a sachet.

Unit Dosage Forms

Accordingly, the present disclosure further provides a unit dosage formcomprising a microorganism (e.g., LAB) of the present disclosure, adried composition of the present disclosure (e.g., a freeze-driedcomposition of the present disclosure), or a pharmaceutical compositionof the present disclosure. In some examples, the unit dosage form is anoral dosage form, such as a tablet, a capsule (e.g., a capsulecontaining a powder or containing micro-pellets or micro-granules), agranule, or a sachet (e.g., containing dried bacteria for suspension ina liquid for oral administration). In some embodiments, thenon-pathogenic microorganism (e.g., LAB) contained in the dosage form isin a dry-powder form or compacted version thereof.

In some examples according to these embodiments, the unit dosage formcontains from about 1×10⁴ to about 1×10¹² colony-forming units (cfu) ofthe microorganism (e.g., LAB). In other examples, the unit dosage formcontains from about 1×10⁶ to about 1×10¹² colony forming units (cfu) ofthe microorganism (e.g., LAB). In other examples, the unit dosage formcontains from about 1×10⁸ to about 1×10¹¹ cfu. In yet other examples,the unit dosage form contains about 1×10⁹ to about 1×10¹² cfu.

Kits

The current disclosure further provides kits containing (1) amicroorganism (e.g., LAB) according to any of the embodiments disclosedherein, a composition containing a microorganism (e.g., LAB) accordingto any of the embodiments described herein, a pharmaceutical compositioncontaining a microorganism (e.g., LAB) according to any of theembodiments described herein, or a unit dosage form containing amicroorganism (e.g., LAB) according to any of the embodiments describedherein; and (2) instructions for administering the microorganism (e.g.,LAB), the composition, the pharmaceutical composition, or the unitdosage form to a mammal, e.g., a human (e.g., human patient).

In related embodiments, the kit may further comprise tests forascertaining the progression of the disease and the success of treatment(e.g., tests for glycemic index, insulin autoantibody (IAA) levels,fragment C levels, and insulitis). The kit may further provide with betacells for transplantation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph demonstrating that an exemplary antigen-specifictherapy according to the present disclosure stably reverses new-onsetdiabetes in NOD mice. New-onset diabetic NOD mice were treated asindicated and blood glucose concentrations were followed up for 14 weekspost-treatment initiation. Shown is the percentage of mice that remaineddiabetic after treatment. “†” indicates dead or moribund mice. In allKaplan-Meier survival curves, statistical significance between groupswas determined by Mantel-Cox log-rank test; *P<0.05, ****, P<0.0001.

FIG. 1B depicts graphs demonstrating that an exemplary antigen-specifictherapy according to the present disclosure preserves residual beta-cellfunction in NOD mice. Intraperitoneal glucose tolerance tests (IPGTTs)were performed on new-onset diabetic NOD mice in addition to L.lactis-based combination therapy (CT)-treated mice (both responders andnon-responders) 1 to 2 weeks prior to treatment termination.Corresponding area under the glucose tolerance curve (AUC glucose;mg/dl×120 minutes) over 2 hours is shown. A responder is used herein asa subject that returns to normoglycemia (e.g. no glucosuria and bloodglucose measurements>200 mg/dl) following treatment.

FIG. 1C is a graph demonstrating that an exemplary antigen-specifictherapy according to the present disclosure halts insulitis progressionin NOD mice. Insulitis scoring was performed in a blinded manner onparaffin-embedded pancreatic sections of new-onset diabetic and L.lactis-based combination therapy (CT)-treated mice (both responders andnon-responders), as indicated, at the end of treatment. Statisticalsignificance between groups was calculated using Mann-Whitney t-test;**P<0.01.

FIG. 2A is a graph showing diabetes remission rate according to startingblood glucose concentrations. New-onset diabetic NOD mice werestratified based on starting glycemia under or above 350 mg/dl at studyentry. Shown is the percentage of mice that remained diabetic aftercombination treatment (CT) with a clinical-grade L. lactis strain of thepresent disclosure. In the Kaplan-Meier survival curve, statisticalsignificance between groups was determined by Mantel-Cox log-rank test;**P<0.01.

FIG. 2B is a graph showing the diabetes remission rate according toinsulin autoantibody (IAA) positivity at study entry. New-onset diabeticNOD mice were stratified based on starting glycemia under or above 350mg/dl at study entry. Shown is the percentage of mice that remaineddiabetic after combination treatment (CT) with a clinical-grade L.lactis strain of the present disclosure.

FIG. 2C is a graph showing that starting glycemia and positivity forIAAs at entry correlated with therapeutic success. New-onset diabeticNOD mice were stratified based on starting glycemia under or above 350mg/dl and IAA positivity at study entry. Shown is the percentage of micethat were tolerized after therapy.

FIG. 2D depicts graphs showing that change in IAA positivity betweenpre- and post-therapy was significantly different in therapy responders.Shown is IAA levels at diabetes diagnosis and after L. lactis-basedcombination treatment follow-up in therapy responders (upper panel) andnon-responders (lower panel). Statistical significance between groupswas calculated using Mann-Whitney t-test; **P<0.01. For one mouse thestarting level of IAA was 139, outside the y-axis limit, and thereforenot shown.

FIG. 3A depicts graphs showing that L. lactis-based combination therapyinduces higher levels of Foxp3⁺ T cells. The percentages of CD25⁺Foxp3⁺cells (left panel), CD25⁻ Foxp3⁺ cells (middle panel), and total Foxp3⁺cells (right panel) within the CD4⁺ T cell population in peripheralblood of new-onset diabetic and L. lactis-based combination therapy(CT)-treated mice (both responders and non-responders). Each symbolrepresents one mouse, and horizontal bars indicate the median value.Statistical significance was calculated using Mann-Whitney t-test;*P<0.05, **P<0.01, ***, P<0.001; ****, P<0.0001.

FIG. 3B depicts graphs showing L. lactis-based combination therapyinduces higher levels of Foxp3⁺ T cells. The percentages of CD25⁺Foxp3⁺cells (left panel), CD25⁻ Foxp3⁺ cells (middle panel), and total Foxp3⁺cells (right panel) within the CD4⁺ T cell population in pancreaticdraining lymph nodes of new-onset diabetic and L. lactis-basedcombination therapy (CT)-treated mice (both responders andnon-responders). Each symbol represents one mouse, and horizontal barsindicate the median value. Statistical significance was calculated usingMann-Whitney t-test; *P<0.05, **P<0.01, ***, P<0.001; ****, P<0.0001.

FIG. 3C depicts graphs showing L. lactis-based combination therapyinduces higher levels of Foxp3⁺ T cells. The percentages of CD25⁺Foxp3⁺cells (left panel), CD25⁻ Foxp3⁺ cells (middle panel), and total Foxp3⁺cells (right panel) within the CD4⁺ T cell population in pancreas ofnew-onset diabetic and L. lactis-based combination therapy (CT)-treatedmice (both responders and non-responders). Each symbol represents onemouse, and horizontal bars indicate the median value. Statisticalsignificance was calculated using Mann-Whitney t-test; *P<0.05,**P<0.01, ***, P<0.001; ****, P<0.0001.

FIG. 3D depicts graphs showing L. lactis-based combination therapyinduces higher levels of Foxp3⁺ T cells. The percentages of CD25Foxp3⁺CTLA4⁺ cells (left panel), CD25⁻Foxp3+CTLA4⁺ cells (middle panel),and Foxp3⁺CTLA4⁺ cells (right panel) within the CD4⁺ T cell populationin peripheral blood of new-onset diabetic and L. lactis-basedcombination therapy (CT)-treated mice (both responders andnon-responders). Each symbol represents one mouse, and horizontal barsindicate the median value. Statistical significance was calculated usingMann-Whitney t-test; *P<0.05, **P<0.01, ***, P<0.001; ****, P<0.0001.

FIG. 3E depicts graphs showing L. lactis-based combination therapyinduces higher levels of Foxp3⁺ T cells. The percentages ofCD25⁺Foxp3⁺CTLA4⁺ cells (left panel), CD25⁻Foxp3+CTLA4⁺ cells (middlepanel), and Foxp3⁺CTLA4⁺ cells (right panel) within the CD4⁺ T cellpopulation in pancreatic draining lymph nodes of new-onset diabetic andL. lactis-based combination therapy (CT)-treated mice (both respondersand non-responders). Each symbol represents one mouse, and horizontalbars indicate the median value. Statistical significance was calculatedusing Mann-Whitney t-test; *P<0.05, **P<0.01, ***, P<0.001; ****,P<0.0001.

FIG. 3F depicts graphs showing L. lactis-based combination therapyinduces higher levels of Foxp3_T cells. The percentages ofCD25⁺Foxp3-CTLA4⁺ cells (left panel), CD25⁻Foxp3+CTLA4⁺ cells (middlepanel), and Foxp3⁺CTLA4⁺ cells (right panel) within the CD4⁺ T cellpopulation in pancreas of new-onset diabetic and L. lactis-basedcombination therapy (CT)-treated mice (both responders andnon-responders). Each symbol represents one mouse, and horizontal barsindicate the median value. Statistical significance was calculated usingMann-Whitney t-test; *P<0.05, **P<0.01, ***, P<0.001; ****, P<0.0001.

FIG. 4 depicts graphs showing that L. lactis-based combination therapyinduces suppressive IL10-secreting Foxp3⁺ T cells in responders andnon-responders. For in vitro polyclonal suppressor assay, CD4⁺CD25⁻effector T cells (Teff) were isolated from normoglycemic NOD mice,dye-labeled, and stimulated for 72 hours using soluble anti-CD3 in thepresence of accessory cells and increasing ratios of CD4⁺CD25⁺Foxp3⁺ orCD4⁺CD25⁻ Foxp3⁺ T cells (Treg), isolated from L. lactis-basedcombination therapy (CT)-treated NOD.Foxp3.hCD2 mice (both respondersand non-responders) at the end of the indicated 6-week treatment.Proliferation of Teff cells was measured by flow cytometric analysis ofdye dilution and shown as the percentage of Teff cells undergone two ormore divisions, normalized to effector only culture. Activation of Teffcells was measured by flow cytometric analysis of CD69 and shown as theMFI, normalized to effector only culture. MSD high-sensitivity multiplexassay of IFN-γ and IL10 concentrations in the Treg:Teff cultures.Statistical significance between groups was calculated usingKruskal-Wallis test followed by Dunnett's multiple testing; *P<0.05,**P<0.01, ***, P<0.001; *P<0.05, **P<0.01.

FIG. 5A is a graph showing L. lactis-based combination therapy-inducedTregs depend on CTLA4 and TGF-β to control T effector cell responses. Teffector (Teff) proliferation—shown as the percentage of Teff cells thathad undergone two or more divisions, normalized to proliferation byeffector only culture. Dye-labeled CD4⁺CD25⁻ T cells (Teff) werestimulated with anti-CD3 (0.5 μg/ml) in the presence of accessory cellsand CD4⁺CD25⁺Foxp3⁺, isolated from L. lactis-based combination therapy(CT)-treated NOD.Foxp3.hCD2 mice (both responders and non-responders),and indicated neutralizing antibodies (10 μg/ml). Statisticalsignificance between groups was calculated using Kruskal-Wallis testfollowed by Dunnett's multiple testing; *P<0.05, **P<0.01, ***P<0.001.

FIG. 5B is a graph showing L. lactis-based combination therapy-inducedTregs depend on CTLA4 and TGF-β to control T effector cell responses. Teffector (Teff) proliferation—shown as the percentage of Teff cells thathad undergone 2 or more divisions, normalized to proliferation byeffector only culture. Dye-labeled CD4⁺CD25⁻ T cells (Teff) werestimulated with anti-CD3 (0.5 μg/ml) in the presence of accessory cellsand CD4⁺CD25⁻ Foxp3⁺ cells (Treg), isolated from L. lactis-basedcombination therapy (CT)-treated NOD.Foxp3.hCD2 mice (both respondersand non-responders), and indicated neutralizing antibodies (10 μg/ml).Statistical significance between groups was calculated usingKruskal-Wallis test followed by Dunnett's multiple testing; *P<0.05,**P<0.01, ***P<0.001.

FIG. 5C is a graph showing that L. lactis-based combinationtherapy-induced Tregs depend on CTLA4 and TGF-β to control T effectorcell responses. L. lactis-based combination therapy-cured mice wereinjected with anti-CTLA4 and anti-TGF-β antibodies (n=5) and followed upfor diabetes recurrence (glucosuria and blood glucose measurements>200mg/dl and).

FIG. 6A is a treatment scheme for the simultaneous administration of L.lactis-based combination therapy (CT) and the specific FOXP3 inhibitorP60 (i.p. 50 μg/daily) in new-onset diabetic NOD mice.

FIG. 6B is a graph showing that specific inhibition of Treg functionimpairs therapy-induced tolerance. Shown is the percentage of mice thatremained diabetic after treatment. In the Kaplan-Meier survival curve,statistical significance between groups was determined by Mantel-Coxlog-rank test; *P<0.05.

FIG. 7A is a treatment scheme for Foxp3⁺ T cell depletion by diphtheriatoxin (DT) in L. lactis-based combination therapy (CT)-curedNOD.Foxp3.DTR mice.

FIG. 7B is a graph showing that Foxp3⁺ T cell depletion breaches L.lactis-based combination therapy-induced tolerance in NOD.Foxp3.DTRmice. Blood glucose measurements in new-onset diabetic NOD.Foxp3.DTRmice during L. lactis-based combination therapy (n=7) and after DTtreatment (n=4). Mice were considered cured (white symbols) when randomblood glucose concentrations recovered to beneath 200 mg/dl or non-cured(black symbols) when mice sustained blood glucose concentrations above200 mg/dl.

FIG. 7C is a graph showing insulitis scoring in the pancreas oftherapy-cured NOD.Foxp3.DTR mice before and after DT treatment.

FIG. 7D is a graph showing the quantification of islet-resident Foxp3⁺ Tcells in the pancreas of therapy-cured NOD.Foxp3.DTR mice before andafter DT treatment. Staining of pancreas sections from combinationtherapy-tolerized mice for insulin (red), CD4 (purple) and Foxp3 (green)in which the white arrow heads indicate the presence of Foxp3⁺ T cellswithin an islet of Langerhans with some insulin positivity. Highermagnification of the boxed area clearly indicates that Foxp3⁺ cells areCD4⁺ T-cells. Statistical significance was calculated using Mann-Whitneyt-test; ****, P<0.0001.

FIG. 8A depicts graphs showing the percentages of CTLA4⁺ cells withinthe CD4⁺CD25⁺Foxp3⁺ (left), CD4⁺CD25⁻Foxp3⁺ (middle), and CD4⁺Foxp3⁺(right) T cell population in peripheral blood of new-onset diabetic andL. lactis-based combination therapy (CT)-treated mice (both respondersand non-responders). Each symbol represents one mouse, and horizontalbars indicate the median value. Statistical significance was calculatedusing Mann-Whitney t-test; *P<0.05, **P<0.01, ***, P<0.001; ****,P<0.0001.

FIG. 8B depicts graphs showing the percentages of CTLA4⁺ cells withinthe CD4⁺CD25⁺Foxp3⁺ (left), CD4⁺CD25⁻Foxp3⁺ (middle), and CD4⁺Foxp3⁺(right) T cell population in pancreatic draining lymph nodes ofnew-onset diabetic and L. lactis-based combination therapy (CT)-treatedmice (both responders and non-responders). Each symbol represents onemouse, and horizontal bars indicate the median value. Statisticalsignificance was calculated using Mann-Whitney t-test; *P<0.05,**P<0.01, ***, P<0.001; ****, P<0.0001.

FIG. 8C depicts graphs showing the percentage of CTLA4⁺ cells within theCD4⁺CD25⁺Foxp3⁺ (left), CD4⁺CD25⁻Foxp3⁺ (middle), and CD4⁺Foxp3⁺ (right)T cell population in pancreas of new-onset diabetic and L. lactis-basedcombination therapy (CT)-treated mice (both responders andnon-responders). Each symbol represents one mouse, and horizontal barsindicate the median value. Statistical significance was calculated usingMann-Whitney t-test; *P<0.05, **P<0.01, ***, P<0.001; ****, P<0.0001.

FIG. 9 is graph showing that L. lactis-based combinationtherapy-tolerized mice are not depleted in pathogenic T effector cells.Adoptive transfer of total splenocytes (1×10⁷) isolated from overtlydiabetic (white diamonds), combination therapy (CT) responders (whitecircles) or non-responders (crossed circles). Statistical calculationswere performed using Mantel-Cox log-rank test; “ns”: not significant.

FIG. 10A is schematic scheme to deplete Foxp3⁺ cells with DT inNOD.Foxp3.DTR mice. Four consecutive i.p. (intraperitoneal) DTinjections (on day (d) 1, 2, 5 and 7) (40 μg/kg body weight/d) areindicated in the scheme.

FIG. 10B depicts graphs showing depletion of Foxp3⁺ cells with DT inNOD.Foxp3.DTR mice. Flow cytometric analysis of peripheral blooddemonstrated efficient depletion of Foxp3⁺ cells in DT-treatedNOD.Foxp3.DTR mice. Foxp3 staining of pancreas sections showed effectivedepletion of islet-resident Foxp3⁺ cells in DT-treated NOD.Foxp3.DTRmice.

FIG. 10C depicts representative flow cytometric profiles showing thepercentage of CD4⁺ T cells positive for Foxp3 and the DTR-GFP fusionprotein before (d0) and after two consecutive DT injections (d3 and 5)of NOD.Foxp3.DTR mice (left panel).

FIG. 10D is a graph demonstrating rapid diabetes onset upon acute Foxp3⁺Treg depletion in NOD.Foxp3.DTR mice.

FIG. 11 is a representation of an exemplary IL-10 expression cassette(SEQ ID NO: 13) comprising an hllA promoter (PhllA), an IL-10 secretionsequence (SSusp45), and an exemplary nucleic acid sequence encodinghuman IL-10 (and a corresponding amino acid sequence), which sequence isused without its native signal peptide and includes a (Pro to Ala) pointmutation at position 2 of the indicated IL-10 sequence (hIL10aPxA). SEQID NO: 14 is the amino acid sequence of the IL-10 secretion sequencelinked to the human IL-10 sequence.

FIGS. 12A and 12B are collectively a representation of an exemplary PINSpolycistronic expression cassette (SEQ ID NO: 15) containing a gapBpromoter (PgapB), the gapB gene, an exemplary intergenic region (rpmD),an exemplary PINS secretion sequence (SSusp45) translationally coupledto an exemplary nucleic acid sequence encoding human PINS. SEQ ID NO: 16is the amino acid sequence encoded by the gapB gene. SEQ ID NO: 17 isthe amino acid sequence encoded by the PINS secretion sequence and thetranslationally coupled human PINS sequence.

FIGS. 13A, 13B, and 13C (SEQ ID NO: 18) are collectively arepresentation of a deletion of the trehalose-6-phosphate phosphorylasegene (trePP; Gene ID: 4797140); Insertion of the constitutive promoterof the HU-like DNA-binding protein gene (PhllA; Gene ID: 4797353) toprecede the putative phosphotransferase genes in the trehalose operon(trePTS; llmg_0453 and llmg_0454; ptsI and ptsII; Gene ID: 4797778 andGene ID: 4797093 respectively), insertion of the intergenic regionpreceding the highly expressed L. lactis MG1363 50S ribosomal proteinL30 gene (rpmD; Gene ID: 4797873) in between ptsI and ptsII.

FIGS. 14A and 14B (SEQ ID NO: 19) are collectively a representation ofinsertion of trehalose-6-phosphate phosphatase gene (otsB; Gene ID:1036914) downstream of unidentified secreted 45-kDa protein gene (usp45;Gene ID: 4797218). Insertion of the intergenic region preceding thehighly expressed L. lactis MG1363 50S ribosomal protein L30 gene (rpmD;Gene ID: 4797873) between usp45 and otsB.

FIGS. 15A and 15B (SEQ ID NO: 20) are collectively a representation ofinsertion of tga at codon position 30 of 446 (tga30), alongside with theintroduction of an EcoRI restriction site, to disrupt the gene encodingcellobiose-specific PTS system IIC component (ptcC; Gene ID: 4796893).

FIG. 16 (SEQ ID NO: 22) is a representation of insertion of a geneencoding a fusion of usp45 secretion leader (SSusp45) with the pinsgene, encoding human proinsulin (PINS; UniProt: P01308, amino acids25-110), downstream of the highly expressed glyceraldehyde 3-phosphatedehydrogenase gene (gapB; Gene ID: 4797877). Insertion of the intergenicregion preceding the highly expressed L. lactis MG1363 50S ribosomalprotein L30 gene (rpmD; Gene ID: 4797873) between gapB and pins.

FIG. 17 (SEQ ID NO: 23) is a representation of deletion of thymidylatesynthase gene (thyA; Gene ID: 4798358). Downstream of the constitutivepromoter of the HU-like DNA-binding protein gene (PhllA; Gene ID:4797353), insertion of a gene encoding a fusion (SEQ ID NO: 24) ofSSusp45 with the hil-10 gene, encoding human interleukin-10 (hIL-10;UniProt: P22301, aa 19-178, variant P2A, Steidler et al., Nat.Biotechnol. 2003, 21(7): 785-789) is inserted to allow expression andsecretion of hIL-10.

FIG. 18: Schematic overview of relevant genetic loci of sAGX0407 asdescribed: trePTS, ΔtrePP; otsB; ptcC-; gapB>>pins and ΔthyA, hIL-10with indication of the relevant oligonucleotide binding sites (oAGXno),EcoRI restriction site, (/truncated/) genetic characters, intergenicregions (IR), PCR amplification product sizes (bp).

DETAILED DESCRIPTION

Provided are compositions and methods for the treatment of T1D, and/orfor restoring tolerance to T1D-specific antigens (i.e., PINS) in asubject.

Definitions

As used in the specification and embodiments, the singular forms “a,”“an” and “the” include plural references unless the context clearlydictates otherwise. For example, the term “a cell” includes a pluralityof cells, including mixtures thereof. Similarly, use of “a compound” fortreatment or preparation of medicaments as described herein contemplatesusing one or more compounds of this invention for such treatment orpreparation unless the context clearly dictates otherwise.

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the recited elements, but not excludingothers. “Consisting essentially of” when used to define compositions andmethods, shall mean excluding other elements of any essentialsignificance to the combination. Thus, a composition consistingessentially of the elements as defined herein would not exclude tracecontaminants from the isolation and purification method andpharmaceutically acceptable carriers, such as phosphate buffered saline,preservatives, and the like. “Consisting of” shall mean excluding morethan trace elements of other ingredients and substantial method stepsfor administering the compositions of this invention. Embodimentsdefined by each of these transition terms are within the scope of thisinvention.

As used herein, the term “expressing” a gene or polypeptide or“producing” a polypeptide (e.g., an IL-10 polypeptide or T1D-specificantigen polypeptide), or “secreting” a polypeptide is meant to include“capable of expressing” and “capable of producing,” or “capable ofsecreting,” respectively. For example, a microorganism, which containsan exogenous nucleic acid can under sufficient conditions (e.g.,sufficient hydration and/or in the presence of nutrients) express andsecrete a polypeptide encoded by an exogenous nucleic acid. However, themicroorganism may not always actively express the encoded polypeptide.The microorganism (e.g., bacterium) may be dried (e.g., freeze-dried),and in that state can be considered dormant (i.e., is not activelyproducing polypeptide). However, once the microorganism is subjected tosufficient conditions, e.g., is administered to a subject and isreleased (e.g., in the gastro-intestinal tract of the subject) it maybegin expressing and secreting polypeptide. Thus, a microorganism“expressing” a gene or polypeptide, “producing” a polypeptide, or“secreting” a polypeptide of the current disclosure includes themicroorganism in its “dormant” state. As used herein, “secrete” meansthat the protein is exported outside the cell and into the culturemedium/supernatant or other extracellular milieu.

As used herein, the term “constitutive” in the context of a promoter (orby extension relating to gene expression or secretion of a polypeptide)refers to a promoter that allows for continual transcription of itsassociated gene. A constitutive promoter compares to an “inducible”promoter.

The term “about” in relation to a reference numerical value, and itsgrammatical equivalents as used herein, can include the referencenumerical value itself and a range of values plus or minus 10% from thatreference numerical value. For example, the term “about 10” includes 10and any amounts from and including 9 to 11. In some cases, the term“about” in relation to a reference numerical value can also include arange of values plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%from that reference numerical value. In some embodiments, “about” inconnection with a number or range measured by a particular methodindicates that the given numerical value includes values determined bythe variability of that method.

An “IL-10 gene” refers to an interleukin 10 gene encoding an “IL-10polypeptide.” The term “IL-10 gene” includes “IL-10 variant genes”encoding “IL-10 variant polypeptides.” The DNA sequence encoding IL-10in an LAB may be codon optimized to facilitate expression in LAB, and assuch may differ from that in the native organism (e.g., humans).

The term “IL-10” or “IL-10 polypeptide” refers to a functional, IL-10polypeptide (e.g., human IL-10 polypeptide) that has at least the aminoacid sequence of the mature form (i.e. without its secretion signal),but also includes membrane-bound forms and soluble forms, as well as“IL-10 variant polypeptides.”

An “IL-10 variant” or “IL-10 variant polypeptide” refers to a modified(e.g., truncated or mutated), but functional IL-10 polypeptide, e.g., atruncated or mutated version of human IL-10. The term “IL-10 variantpolypeptide” includes IL-10 polypeptides with enhanced activity ordiminished activity when compared to a corresponding wild-type IL-10polypeptide. An “IL-10 variant polypeptide” retains at least some IL-10activity (functional polypeptide).

The “percentage identity” between polypeptide sequences can becalculated using commercially available algorithms which compare areference sequence with a query sequence. In some embodiments,polypeptides are 70%, at least 70%, 75%, at least 75%, 80%, at least80%, 85%, at least 85%, 90%, at least 90%, 92%, at least 92%, 95%, atleast 95%, 97%, at least 97%, 98%, at least 98%, 99%, or at least 99% or100% identical to a reference polypeptide, or a fragment thereof (e.g.,as measured by BLASTP or CLUSTAL, or other alignment software) usingdefault parameters. Similarly, nucleic acids can also be described withreference to a starting nucleic acid, e.g., they can be 50%, at least50%, 60%, at least 60%, 70%, at least 70%, 75%, at least 75%, 80%, atleast 80%, 85%, at least 85%, 90%, at least 90%, 95%, at least 95%, 97%,at least 97%, 98%, at least 98%, 99%, at least 99%, or 100% identical toa reference nucleic acid or a fragment thereof (e.g., as measured byBLASTN or CLUSTAL, or other alignment software using defaultparameters). When one molecule is said to have a certain percentage ofsequence identity with a larger molecule, it means that when the twomolecules are optimally aligned, the percentage of residues in thesmaller molecule finds a match residue in the larger molecule inaccordance with the order by which the two molecules are optimallyaligned, and the “%” (percent) identity is calculated in accord with thelength of the smaller molecule.

The term “chromosomally integrated” or “integrated into a chromosome” orany variation thereof means that a nucleic acid sequence (e.g., gene;open reading frame; exogenous nucleic acid encoding a polypeptide;promoter; expression cassette; and the like) is located on (integratedinto) a microbial (e.g., bacterial) chromosome, i.e., is not located onan episomal vector, such as a plasmid. In some embodiments, in which thenucleic acid sequence is chromosomally integrated, the polypeptideencoded by such chromosomally integrated nucleic acid is constitutivelyexpressed.

The terms “self-antigen” or “auto-antigen” are used interchangeablyherein. The terms are used herein in accordance with the art recognizedmeaning of self-antigen or auto-antigen, and generally refer to apolypeptide/protein originating from within a subjects own body(produced by the subject's own body), wherein the antigen is recognizedby the subject's own immune system, and typically produces antibodiesagainst such antigen. Autoimmune diseases are generally associated withcertain disease-specific self-antigens. For example, in T1D a subject'simmune system may produce antibodies against at least one antigenassociated with the beta-cell destruction process. Such self-antigensinclude proinsulin (PINS), glutamic acid decarboxylase (GAD65),insulinoma-associated protein 2 (IA-2), islet-specificglucose-6-phosphatase catalytic subunit-related protein (IGRP) and zinctransporter (ZnT) 8. Clinical T1D may further be associated withadditional candidate target molecules expressed by beta-cells such aschromogranin A, (prepro) islet amyloid polypeptide (ppIAPP), peripherinand citrullinated glucose-regulated protein (GRP).

The term “T1D-specific antigen gene” refers to a gene encoding the above“T1D-specific antigen.” The term “T1D-specific antigen gene” includes“T1D-specific antigen variant genes” encoding “T1D-specific antigenvariant polypeptides.” The DNA sequence encoding T1D antigen in an LABmay be codon optimized to facilitate expression in LAB, and as such maydiffer from that in the native organism (e.g. humans).

The term “T1D-specific antigen polypeptide” refers to a functional,e.g., full-length, polypeptide, as well as “T1D-specific antigen variantpolypeptides,” which may have enhanced activity or diminished activitywhen compared to a corresponding wild-type polypeptide The T1D specificantigen may also lack the eukaryotic signal sequences.

The term “T1D-specific antigen variant” or “T1D-specific antigen variantpolypeptide” refers to a modified (e.g., truncated or mutated), butfunctional polypeptide, e.g., a truncated or mutated version of humanPINS. The term “variant polypeptide” includes polypeptides with enhancedactivity or diminished activity when compared to a correspondingwild-type polypeptide. A “variant polypeptide” retains at least somebiological activity (functional polypeptide). Exemplary variants ofGAD65 and IA-2 include trimmed versions thereof (e.g., GAD65₃₇₀₋₅₇₅, andIA-2₆₃₅₋₉₇₉, respectively; relative to NCBI accession numbersNP_000809.1 and NP_002837.1 retaining antigenic properties, and are thususeful in the compositions and methods of the current disclosure, e.g.,in stimulating Tregs and inducing tolerance in a subject. Generally,trimmed or truncated versions of a T1D-specific antigen are efficientlyexpressed and secreted by the microorganism (e.g., Lactococcus lactis).

The term “operably linked” refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner. A control sequence “operably linked” to acoding sequence is ligated in such a way that expression of the codingsequence is achieved under condition compatible with the controlsequences. For example, a promoter is said to be operably linked to agene, open reading frame or coding sequence, if the linkage orconnection allows or effects transcription of said gene. In a furtherexample, a 5′ and a 3′ gene, cistron, open reading frame or codingsequence are said to be operably linked in a polycistronic expressionunit, if the linkage or connection allows or effects translation of atleast the 3′ gene. For example, DNA sequences, such as, e.g., a promoterand an open reading frame, are said to be operably linked if the natureof the linkage between the sequences does not (1) result in theintroduction of a frame-shift mutation, (2) interfere with the abilityof the promoter to direct the transcription of the open reading frame,or (3) interfere with the ability of the open reading frame to betranscribed by the promoter region sequence.

Anti-CD3 Antibody

An “anti-CD3 antibody” can be any antibody that binds to a CD3 receptoron the surface of a T cell, e.g., an antibody that targets the epsilonchain of CD3 (CD3E). The term “anti-CD3 antibody” includes any fragmentof such antibody, such as Fab fragments, single domain antibodies,nanobodies and the like. In some examples, the anti-CD3 antibody orfragment thereof is a monoclonal antibody. In other examples, theanti-CD3 antibody is a humanized monoclonal antibody, e.g., a chimericor humanized hybrid antibody. In some examples, the anti-CD3 antibody isa single domain antibody or nanobody. In other examples, the anti-CD3antibody is muromonab-CD3. Other known monoclonal anti-CD3 antibodiesinclude otelixizumab, teplizumab and visilizumab, and fragments thereof.These antibodies are being investigated for the treatment of conditionslike Crohn's disease, ulcerative colitis and type 1 diabetes (see, e.g.,Herold K. C. and Taylor L.; “Treatment of Type 1 diabetes with anti-CD3monoclonal antibody: induction of immune regulation?”. ImmunologicResearch 2003, 28 (2): 141-50) and for inducing immune tolerance. See,e.g., Bisikirska et al. “TCR stimulation with modified anti-CD3 mAbexpands CD8+ T cell population and induces CD8+CD25+Tregs” (2005), andBisikirska et al., “Use of Anti-CD3 Monoclonal Antibody to Induce ImmuneRegulation in Type 1 Diabetes”. Annals of the New York Academy ofSciences 2004, 1037: 1-9. In some examples, the anti-CD3 antibody blocksthe function of effector T cells (e.g., which attack and destroyinsulin-producing beta-cells) and/or stimulates regulatory T cells.Thus, in some examples, the anti-CD3 antibody protects a subject againsteffector T cell damage, e.g., preserving the ability of beta-cells toproduce insulin. In some examples, the anti-CD3 antibody is suitable foradministration to a human, e.g., is the subject of a clinical trial orhas already been approved by a regulatory agency. In some examples, theanti-CD3 antibody is otelixizumab or teplizumab.

Recent-Onset T1D

The inventors have discovered that subjects with certain minimal amountsof residual beta-cell function respond particularly well to thetherapeutic methods described herein. Residual beta-cell function may bemeasured in accordance with art recognized methods and as describedherein. However, sufficient residual beta-cell function is typicallyfound in subjects who have not been exposed to harmful auto-immunefunctions targeting the subject's beta-cells for too long. Thus, in someembodiments, the mammalian subject in the above methods has recentlybeen diagnosed with T1D. For example, such subject may be referred to ashaving “recent-onset T1D” or “new-onset T1D” (used interchangeablyherein). In some examples according to these embodiments, the subjecthas been diagnosed with T1D within the previous 12 months, the previous18 months, or the previous 24 months prior to administering acomposition according to the present disclosure. In other examples, thesubject has been treated with insulin for less than about 12 weeks, lessthan about 8 weeks, less than about 6 weeks, or less than about 4 weeks.In other examples, according to any of the above embodiments, thesubject tested positive for at least one auto-antibody, e.g., testedpositive for insulin autoantibody (IAA), islet-cell auto-antibodies,glutamic acid decarboxylase (e.g., GAD65) auto-antibodies, and/or ICA512antibodies. In other examples, according to any of the aboveembodiments, the subject has a fasting plasma glucose level of greaterthan about 100 mg/dL, or greater than about 120 mg/dL, or greater thanabout 126 mg/dL. In other examples, according to any of the aboveembodiments, the subject has a fasting plasma glucose level of about 180to about 250 mg per deciliter (mg/dL), or about 10 mmol/L to about 14mmol/L. In other examples, according to any of the above embodiments,the subject has a plasma glucose level of about 100 to about 500 mg/dL,In other examples, according to any of the above embodiments, thesubject had polyuria for less than about 8 months, less than about 6months, or less than about 4 months.

In some examples, a human subject may be categorized as having earlyonset T1D when any one of the following is measured in the subject: (1)two consecutive fasting blood glucose tests are equal to or greater than126 mg/dL; (2) when any random blood glucose measurement is greater than200 mg/dL; (3) a hemoglobin Alc (HbA1c) test that is equal to or greaterthan 6.5 percent (HbA1c is a blood test that gives a three month averageof blood sugars); or (4) a two-hour oral glucose tolerance test with anyvalue over 200 mg/dL, or combinations thereof. Thus, normal glycemia maybe characterized by any of the following: (1) two consecutive fastingblood glucose tests are below 126 mg/dL, below 120 mg/dL, or below 100mg/dL; (2) when any random blood glucose measurement is below 200 mg/dL,below 180 mg/dL, below 160 mg/dL, or below 140 mg/dL; (3) a hemoglobinAlc (HbA1c) test that is less than 6.5 percent, or less than 6%; or (4)a two-hour oral glucose tolerance test with a value below 200 mg/dL,below 180 mg/dL, below 160 mg/dL, or below 140 mg/dL, or any combinationthereof.

A subject that returns to normoglycemia (e.g. no glucosuria and bloodglucose measurements>200 mg/dl) following treatment may be considered tobe a “responder” to the treatment. Subject who do not return tosubstantial normoglycemia are “non-responders”.

The concentrations of C-peptide in the blood or urine of a subject maybe used to assess a subject's beta-cell function and the T1D diseasestage, where higher concentrations of C-peptide indicate higherbeta-cell function. In some examples, the subject (e.g., human patient)has recent-onset T1D, characterized by having a fasting blood C-peptideconcentration of less than about 1 nmol/L, but at least about 0.2nmol/L. In other embodiments, the recent-onset subject (e.g., humanpatient) has a stimulated blood C-peptide concentration, e.g., during a4-hour mixed meal tolerance test (MMTT) of less than about 5 nmol/L, butat least about 0.2 nmol/L, or less than about 4 nmol/L, but at leastabout 0.5 nmol/L. Other suitable C-peptide ranges are described herein.Stimulated C-peptide may thus be measured using MMTT area under thecurve C-peptide (AUC CP), or may alternatively be measured using a 90minute MMTT stimulated CP (90CP post MMTT or 90CP).

Promoter

By “promoter” is meant generally a region on a nucleic acid molecule,for example DNA molecule, to which an RNA polymerase binds and initiatestranscription. A promoter is for example, positioned upstream, i.e., 5′,of the sequence the transcription of which it controls. The skilledperson will appreciate that the promoter may be associated withadditional native regulatory sequences or regions, e.g. operators. Theprecise nature of the regulatory regions needed for expression may varyfrom organism to organism, but shall in general include a promoterregion which, in prokaryotes, contains both the promoter (which directsthe initiation of RNA transcription) as well as the DNA sequences which,when transcribed into RNA, will signal the initiation of proteinsynthesis. Such regions will normally include those 5′-non-codingsequences involved with initiation of transcription and translation,such as the Pribnow-box (cf. TATA-box), Shine-Dalgarno sequence, and thelike.

Expression Cassette

The term “expression cassette” or “expression unit” is used inaccordance with its generally accepted meaning in the art, and refers toa nucleic acid containing one or more genes and sequences controllingthe expression of the one or more genes. Exemplary expression cassettescontain at least one promoter sequence and at least one open readingframe.

Polycistronic Expression Cassette

The terms “polycistronic expression cassette” “polycistronic expressionunit” or “polycistronic expression system” are used hereininterchangeably and in accordance with their generally accepted meaningin the art. They refer to a nucleic acid sequence wherein the expressionof two or more genes is regulated by common regulatory mechanisms, suchas promoters, operators, and the like. The term polycistronic expressionunit as used herein is synonymous with multicistronic expression unit.Examples of polycistronic expression units are without limitationbicistronic, tricistronic, tetracistronic expression units. Any mRNAcomprising two or more, such as 3, 4, 5, 6, 7, 8, 9, 10, or more, openreading frames or coding regions encoding individual expression productssuch as proteins, polypeptides and/or peptides is encompassed within theterm polycistronic. A polycistronic expression cassette includes atleast one promoter, and at least two open reading frames controlled bythe promoter, wherein an intergenic region is optionally placed betweenthe two open reading frames.

In some example, the “polycistronic expression cassette” includes one ormore endogenous genes and one or more exogenous genes that aretranscriptionally controlled by a promoter which is endogenous to themicroorganism (e.g., LAB). The polycistronic expression unit or systemas described herein can be transcriptionally controlled by a promoterthat is exogenous to the microorganism (e.g., LAB). In a furtherembodiment, the translationally or transcriptionally coupled one or moreendogenous genes and one or more exogenous genes as described herein aretranscriptionally controlled by the native promoter of (one of) said oneor more endogenous genes. In another embodiment, the polycistronicexpression unit is transcriptionally controlled by the native promoterof (one of) said one or more endogenous genes comprised in saidpolycistronic expression unit. In another embodiment, the polycistronicexpression unit is operably linked to a gram-positive endogenouspromoter. In an exemplary embodiment, the promoter may be positionedupstream of, i.e., 5′ of the open reading frame(s) to which it isoperably linked. In a further embodiment, the promoter is the nativepromoter of the 5′ most, i.e., most upstream, endogenous gene in thepolycistronic expression unit. Accordingly, in some examples, thepolycistronic expression unit contains an endogenous gene and one ormore exogenous genes transcriptionally coupled to the 3′ end of said oneor more endogenous gene, for example wherein said one or more exogenousgene(s) is (are) the most 3′ gene(s) of the polycistronic expressionunit.

As used herein, the term “translationally coupled” is synonymous with“translationally linked” or “translationally connected”. These terms inessence relate to polycistronic expression cassettes or units. Two ormore genes, open reading frames or coding sequences are said to betranslationally coupled when common regulatory element(s) such as inparticular a common promoter effects the transcription of said two ormore genes as one mRNA encoding said two or more genes, open readingframes or coding sequences, which can be subsequently translated intotwo or more individual polypeptide sequences. The skilled person willappreciate that bacterial operons are naturally occurring polycistronicexpression systems or units in which two or more genes aretranslationally or transcriptionally coupled.

Intergenic Region

As used herein, the term “intergenic region” is synonymous with“intergenic linker” or “intergenic spacer”. An intergenic region isdefined as a polynucleic acid sequence between adjacent (i.e., locatedon the same polynucleic acid sequence) genes, open reading frames,cistrons or coding sequences. By extension, the intergenic region caninclude the stop codon of the 5′ gene and/or the start codon of the 3′gene which are linked by said intergenic region. As defined herein, theterm intergenic region specifically relates to intergenic regionsbetween adjacent genes in a polycistronic expression unit. For example,an intergenic region as defined herein can be found between adjacentgenes in an operon. Accordingly, in an embodiment, the intergenic regionas defined herein is an operon intergenic region.

In some examples, the intergenic region, linker or spacer is selectedfrom intergenic regions preceding, i.e., 5′ to, more particularlyimmediately 5′ to, rp/W, rpf P, rpmD, rp/8, rpsG, rpsE or rp/N of agram-positive bacterium. In an embodiment, said gram positive bacteriumis a lactic acid bacterium, for example a Lactococcus species, e.g.,Lactococcus lactis, and any subspecies or strain thereof. In anembodiment, said intergenic region encompasses the start codon of rp/W,rp/P, rpmD, rp/8, rpsG, rpsE or rp/N and/or the stop codon of thepreceding, i.e. 5′, gene. In a preferred embodiment, the inventionrelates to a gram-positive bacterium or a recombinant nucleic acid asdescribed herein, wherein the endogenous gene and the one or moreexogenous genes are transcriptionally coupled by intergenic region orregions active in the gram-positive bacterium, for example wherein theintergenic region or regions is endogenous to said gram-positivebacterium, for example, wherein the endogenous intergenic region isselected from intergenic regions preceding rp/W, rpf P, rpmD, rp/8,rpsG, rpsE, rp/N, rplM, rplE, and rplF.

The skilled person will appreciate that if the intergenic regionencompasses a 5′ stop codon and/or a 3′ start codon, these respectivecodons in some cases are not present in the genes which are linked bysaid intergenic regions, in order to avoid double start and/or stopcodons, which may affect correct translation initiation and/ortermination. Methods for identifying intergenic regions are known in theart. By means of further guidance, intergenic regions can for instancebe identified based on prediction of operons, and associated promotersand open reading frames, for which software is known and available inthe art. Exemplary intergenic regions are described in internationalpatent application publication WO2012/164083, the disclosure of which isincorporated herein by reference in its entirety.

Subject

A “subject” is an organism, which may benefit from being administered acomposition of the present disclosure, e.g., according to methods of thepresent disclosure. The subject may be a mammal (“mammalian subject”).Exemplary mammalian subjects include humans, farm animals (such as cows,pigs, horses, sheep, goats), pets or domesticated animals (such as adogs, cats, and rabbits), and other animals, such as mice, rats, andprimates. In some examples, the mammalian subject is a human patient.

The term “international unit” (IU) is used herein in accordance with itsart-recognized meaning and represents an amount of a substance (e.g.,polypeptide). The mass or volume that constitutes one international unitvaries based on which substance is being measured. The World HealthOrganization (WHO) provides unit characterizations for bioactivepolypeptides.

T1D Specific Antigen

The at least one microorganism of the present disclosure contains anexogenous nucleic acid encoding at least one disease-specific (i.e.,T1D-specific) self-antigen gene, and can express such gene underconditions sufficient for expression. Exemplary T1D-specificself-antigens include islet antigens associated with the beta-celldestruction process. In some embodiments, in any of the abovecompositions and methods, the T1D-specific antigen is selected fromknown auto-antigens associated with T1D, and include proinsulin (PINS);insulin (INS); glutamic acid decarboxylase (GAD) (e.g., GAD65, GAD67, orGAD2); insulinoma-associated protein 2 (islet antigen-2; IA-2) (alsoreferred to as protein tyrosine phosphatase, receptor type, N (PTPRN),tyrosine phosphatase-like protein, or ICA512), (see, e.g., Long et al.,Diabetes 2013, 62 (6), 2067-2071); islet-specific glucose-6-phosphatasecatalytic subunit-related protein (IGRP), zinc transporter 8 (ZnT8).Other examples include molecules expressed by beta-cells, such aschromogranin A, (prepro) islet amyloid polypeptide (ppIAPP), peripherin,citrullinated glucose-regulated protein (e.g., GRP78); see, e.g., Rondaset al., Diabetes 2015; 64(2):573-586; and Ye et al., Diabetes 2010,59(1):6-16), and combinations of two or more of these antigens. In otherembodiments, in the above compositions and methods, the T1D-specificantigen is PINS, GAD65, or IA-2. In other embodiments, in the abovecompositions and methods, the T1D-specific antigen is PINS. In variousembodiments, the T1D-specific antigen is encoded by a variant nucleicacid sequence shorter than a full length (e.g., wild-type) gene, as such“trimmed” versions are often more efficiently expressed and/or secretedby the microorganisms used (e.g., Lactococcus lactis). While secretionis more efficient, many “trimmed” versions retain all (or a substantialportion) of their biological activity, e.g., retain sufficient Tregstimulating and/or tolerance-inducing capacities.

Examples of PINS polypeptides include wild-type human PINS andpolypeptides having at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, or at least about 99% sequenceidentity with such wild-type human PINS. An exemplary amino acidsequence of wild-type human PINS is SEQ ID NO: 5, and an exemplarynucleic acid sequence is represented by SEQ ID NO: 6 (see CDS containedin accession number NM_000207.2). In some examples, the PINS polypeptidehas an amino acid sequence of SEQ ID NO: 3, which is encoded by anucleic acid sequence of SEQ ID NO: 4.

Additional exemplary PINS nucleotide sequences are represented by thecoding sequences of NCBI accession numbers AY899304 (complete CDS,alternatively spliced; SEQ ID NO: 7); NM_000207 (transcript variant 1);NM_001185097 (transcript variant 2); NM_001185098 (transcript variant3); NM_001291897 (transcript variant 4), and partial sequences thereof.Exemplary PINS amino acid sequences include those encoded by any one ofthe above PINS nucleic acid sequences.

Any nucleotide sequence encoding the amino acid sequence of SEQ ID NO:3, or any nucleotide sequence encoding at least about 20, at least about30, at least about 40, at least about 50, at least about 60, at leastabout 70, or at least about 80 consecutive amino acids thereof, or anynucleotide sequence encoding a polypeptide having at least about 60%, atleast about 70%, at least about 80%, at least about 90%, at least about95%, at least about 96%, at least about 97%, at least about 98%, or atleast about 99% sequence identity with SEQ ID NO: 3 may be used.

Additional PINS polypeptides are described, e.g., in UniProtKB-P01308and links therein. In some examples, the PINS polypeptide is representedby amino acid residues 25-110 (numbering according to SEQ ID NO: 5).

Exemplary GAD (e.g., GAD65) polypeptides include wild-type human GAD65,and polypeptides having at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, or at least about 99% sequenceidentity with such wild-type GAD65. See, e.g., CDS contained inaccession number M81882.1.

Any nucleotide sequence encoding the above amino acid sequence, or anynucleotide sequence encoding at least about 100, at least about 200, atleast about 300, at least about 400, or at least about 500 consecutiveamino acids thereof, or any nucleotide sequence encoding a polypeptidehaving at least about 60%, at least about 70%, at least about 80%, atleast about 90%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% sequence identity may beused.

Other exemplary glutamate decarboxylase (e.g., GAD65) sequences aredescribed, e.g., in UniProtKB-Q05329 and links therein. In some example,the GAD polypeptide is a trimmed variant containing less than about 500,less than about 400, or less than about 300 of the wild-type aminoacids. Exemplary polypeptide fragments (trimmed GAD65 variants) aredescribed, e.g., in Robert et al., Benef Microbes 2015, 6(4): 591-601,the disclosure of which is incorporated herein by reference in itsentirety. In some examples, the trimmed GAD variants are efficientlyexpressed and secreted by a gram-positive bacterium (i.e., Lactococcuslactis). An exemplary trimmed GAD variant is GAD65₃₇₀-5₇₅ (amino acidnumbering relative to NCBI accession number NP 000809.1).

Other exemplary GAD nucleotide sequences are represented by NCBIaccession numbers M81882 (GAD65); M81883 (GAD67); NM_000818 (GAD2variant 1); and NM_001134366 (GAD2 variant 2); and open reading frames(CDS) contained therein. Exemplary amino acid sequences includesequences encoded by the above nucleotide sequences of accession numbersM81882, M81883, NM_001134366, and NM 000818.

A person of ordinary skill in the art will appreciate that the optimalamount of self-antigen to be delivered to the subject using the methodsof the present disclosure varies, e.g., with the type of antigen, themicroorganism expressing the antigen, and the genetic construct, e.g.,the strength of the promoter used in the genetic construct. Typically,the microorganism will be administered in an amount equivalent to aparticular amount of expressed antigen, or in an amount, which generatesa desired PK profile for the respective antigen polypeptide in therespective subject. Exemplary daily antigen doses are from about 10 fgto about 100 μg of active polypeptide per day. Other exemplary doseranges are from about 1 μg to about 100 μg per day; or from about 1 ngto about 100 μg per day.

The above antigen doses may be realized by administering to the subjecteffective amounts of the microorganism per day, wherein themicroorganism is adapted to express a sufficient amount of bioactivepolypeptide to realize the desired dose, such as those above. Themicroorganism secreting the antigen polypeptide may be delivered in adose of from about 10⁴ colony forming units (cfu) to about 10¹² cfu perday, e.g., from about 10⁶ cfu to about 10¹² cfu per day, or from about10⁹ cfu to about 10¹² cfu per day.

The amount of secreted antigen polypeptide can be determined based oncfu, for example in accordance with the methods described in Steidler etal., Science 2000; 289(5483):1352-1355, or by using ELISA. For example,a particular microorganism may secrete at least about 1 ng to about 1 μgof active polypeptide per 10⁹ cfu. Based thereon, the skilled person cancalculate the range of antigen polypeptide secreted at other cfu doses.

Each of the above doses/dose ranges may be administered in connectionwith any dosing regimen as described herein. The daily dose of activepolypeptide may be administered in 1, 2, 3, 4, 5, or 6 portionsthroughout the day. Further the daily doses may be administered for anynumber of days, with any number of rest periods between administrationperiods. For example, a dose of from about 0.01 to about 3.0 M IU ofIL-10/day/subject may be administered every other day for a total of 6weeks.

Treating

The terms “treatment”, “treating”, and the like, as used herein meansameliorating or alleviating characteristic symptoms or manifestations ofa disease or condition, e.g., T1D. For example, treatment of T1D canresult in the restoration or induction of antigen-specific immunetolerance in the subject. In other examples, treatment means arrestingauto-immune diabetes, or reversing autoimmune diabetes. As used hereinthese terms also encompass, preventing or delaying the onset of adisease or condition or of symptoms associated with a disease orcondition, including reducing the severity of a disease or condition orsymptoms associated therewith prior to affliction with said disease orcondition. Such prevention or reduction prior to affliction refers toadministration of the compound or composition of the invention to apatient that is not at the time of administration afflicted with thedisease or condition. “Preventing” also encompasses preventing therecurrence or relapse-prevention of a disease or condition or ofsymptoms associated therewith, for instance after a period ofimprovement. Treatment of a subject “in need thereof” conveys that thesubject has a diseases or condition, and the therapeutic method of theinvention is performed with the intentional purpose of treating thespecific disease or condition.

Patient Sub-Populations

In some embodiments, the subject being treated using the methods of thepresent disclosure, has significant (e.g., measurable) residualbeta-cell function. Under such circumstances, the subject may maintaindisease remission, even after treatment is interrupted or stoppedaltogether. Newly diagnosed patients often have a certain minimal numberof pancreatic islet beta-cells (beta-cells) remaining at the time ofdiagnosis, so that such patients are able to produce a certain minimalamount of endogenous insulin. Such patient population can benefitparticularly well when treated with the compositions and methods of thecurrent disclosure (e.g., IL-10 and PINS therapy). The treatmentsdescribed herein can prevent further destruction of beta-cells and maythus induce disease remission. Unexpectedly, the inventors have foundthat initial beta-cell mass can be important for the efficacy oftreatment. However, once a subject's beta-cells are destroyed, suchsubject may no longer benefit from the described treatment in the samemanner.

Untreated T1D develops over time and becomes progressively worse as betacells in the pancreas are destroyed. Thus, as the present method canhalt disease, it is advantageous to treat subjects as early as possiblein the disease progression. Thus, in a related embodiment, thetherapeutic method of the invention also includes measuring diseaseprogression prior to therapy, and during therapy.

Therapeutically Effective Amount

As used herein, the term “therapeutically effective amount” refers to anamount of a non-pathogenic microorganism or a composition of the presentdisclosure that will elicit a desired therapeutic effect or responsewhen administered according to the desired treatment regimen. In somecases, the compounds or compositions are provided in a unit dosage form,for example a tablet or capsule, which contains an amount of the activecomponent equivalent with the therapeutically effective amount whenadministered once, or multiple times per day.

A person of ordinary skill in the art will appreciate that atherapeutically effective amount of a recombinant microorganism, whichis required to achieve a desired therapeutic effect (e.g., for theeffective treatment of T1D), will vary, e.g., depending on the nature ofthe IL-10 polypeptide expressed by the microorganism, the nature of theantigen polypeptide expressed by the microorganism, the route ofadministration, and the age, weight, and other characteristics of therecipient.

Mucosa

The term “mucosa” or “mucous membrane” is used herein in accordance withits art recognized meaning. The “mucosa” can be any mucosa found in thebody, such as oral mucosa, rectal mucosa, gastric mucosa, intestinalmucosa, urethral mucosa, vaginal mucosa, ocular mucosa, buccal mucosa,bronchial or pulmonary mucosa, and nasal or olfactory mucosa.

The term “mucosal delivery” as used herein is used in accordance withits art recognized meaning, i.e., delivery to the mucosa, e.g., viacontacting a composition of the present disclosure with a mucosa. Oralmucosal delivery includes buccal, sublingual and gingival routes ofdelivery. Accordingly, in some embodiments, “mucosal delivery” includesgastric delivery, intestinal delivery, rectal delivery, buccal delivery,pulmonary delivery, ocular delivery, nasal delivery, vaginal deliveryand oral delivery.

The term “mucosal tolerance” refers to the inhibition of specific immuneresponsiveness to an antigen in a mammalian subject (e.g., a humanpatient), after the subject has been exposed to the antigen via themucosal route. In some cases the mucosal tolerance is systemictolerance. Low dose oral tolerance is oral tolerance induced by lowdoses of antigens, and is characterized by active immune suppression,mediated by cyclophosphamide sensitive regulatory T-cells that cantransfer tolerance to naive hosts. High dose oral tolerance is oraltolerance induced by high doses of antigens, is insensitive tocyclophosphamide treatment, and proceeds to induction of T cellhyporesponsiveness via anergy and/or deletion of antigen specificT-cells. The difference in sensitivity to cyclophosphamide can be usedto make a distinction between low dose and high dose tolerance (Strobelet al., 1983). In some cases, the oral tolerance is low dose oraltolerance as described by Mayer and Shao (2004).

Immuno-Modulating Compound

The terms “immuno-modulating compound” or immuno-modulator” are usedherein in accordance with their art-recognized meaning. Theimmuno-modulating compound can be any immuno-modulating compound knownto a person skilled in the art.

In some embodiments, the immuno-modulating compound is a toleranceinducing compound. Tolerance induction can be obtained, e.g., byinducing regulatory T-cells, or in an indirect way, e.g., by activationof immature dendritic cells to tolerizing dendritic cells and/orinhibiting Th2 immune response inducing expression of “co-stimulation”factors on mature dendritic cells. Immuno-modulating andimmuno-suppressing compounds are known to the person skilled in the artand include, but are not limited to, bacterial metabolites such asspergualin, fungal and streptomycal metabolites such as tacrolimus orciclosporin, immuno-suppressing cytokines such as IL-4, IL-10, IFNα,TGFβ (as selective adjuvant for regulatory T-cells) Flt3L, TSLP andRank-L (as selective tolerogenic DC inducers), antibodies and/orantagonist such as anti-CD40L, anti-CD25, anti-CD20, anti-IgE, anti-CD3,and proteins, peptides or fusion proteins such as the CTL-41 g or CTLA-4agonist fusion protein. In some embodiments, the immuno-modulatingcompound is an immuno-suppressing compound. In other embodiments, theimmuno-suppressing compound is an immuno-suppressing cytokine orantibody. In other embodiments, the immuno-suppressing cytokine is atolerance-enhancing cytokine or antibody. It will be appreciated by theperson skilled in the art that the term “immuno-modulating compound”also includes functional homologues thereof. A functional homologue is amolecule having essentially the same or similar function for theintended purposes, but can differ structurally. In some examples, theimmuno-modulating compound is anti-CD3, or a functional homologuethereof.

Microorganisms

The invention relates to the use of at least one microorganism. In someembodiments, the microorganism is a non-pathogenic and non-invasivebacterium. In other embodiments, the microorganism is a non-pathogenicand non-invasive yeast.

In some embodiments, the microorganism is a yeast strain selected fromthe group consisting of Saccharomyces sp., Hansenula sp., Kluyveromycessp. Schizzosaccharomyces sp. Zygosaccharomyces sp., Pichia sp., Monascussp., Geothchum sp and Yarrowia sp. In some embodiments, the yeast isSaccharomyces cerevisiae. In other embodiments, the S. cerevisiae is ofthe subspecies boulardii. In one embodiment of the present invention,the recombinant yeast host-vector system is a biologically containedsystem. Biological containment is known to the person skilled in the artand can be realized by the introduction of an auxotrophic mutation, forexample a suicidal auxotrophic mutation such as the ThyA mutation, orits equivalents.

In other embodiments of the present invention, the microorganism is abacterium, such as a non-pathogenic bacterium, e.g., a food gradebacterial strain. In some examples, the non-pathogenic bacterium is agram-positive bacterium, e.g., a gram-positive food-grade bacterialstrain. In some embodiments, the gram-positive food grade bacterialstrain is a lactic acid fermenting bacterial strain (i.e., a lactic acidbacterium (LAB) or a Bifidobacterium).

In some embodiments, the lactic acid fermenting bacterial strain is aLactococcus, Lactobacillus or Bifidobacterium species. As used herein,Lactococcus or Lactobacillus is not limited to a particular species orsubspecies, but meant to include any of the Lactococcus or Lactobacillusspecies or subspecies. Exemplary Lactococcus species include Lactococcusgarvieae, Lactococcus lactis, Lactococcus piscium, Lactococcusplantarum, and Lactococcus raffinolactis. In some examples, theLactococcus lactis is Lactococcus lactis subsp. cremoris, Lactococcuslactis subsp. hordniae, or Lactococcus lactis subsp. Lactis.

Exemplary Lactobacillus species include Lactobacillus acetotolerans,Lactobacillus acidophilus, Lactobacillus agilis, Lactobacillus algidus,Lactobacillus alimentarius, Lactobacillus amylolyticus, Lactobacillusamylophilus, Lactobacillus amylovorus, Lactobacillus animalis,Lactobacillus aviarius, Lactobacillus aviarius subsp. araffinosus,Lactobacillus aviarius subsp. aviarius, Lactobacillus bavaricus,Lactobacillus bifermentans, Lactobacillus brevis, Lactobacillusbuchneri, Lactobacillus bulgaricus, Lactobacillus carnis, Lactobacilluscasei, Lactobacillus casei subsp. alactosus, Lactobacillus casei subsp.casei, Lactobacillus casei subsp. pseudoplantarum, Lactobacillus caseisubsp. rhamnosus, Lactobacillus casei subsp. tolerans, Lactobacilluscatenaformis, Lactobacillus cellobiosus, Lactobacillus collinoides,Lactobacillus confusus, Lactobacillus coryniformis, Lactobacilluscoryniformis subsp. coryniformis, Lactobacillus coryniformis subsp.torquens, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacilluscurvatus subsp. curvatus, Lactobacillus curvatus subsp. melibiosus,Lactobacillus delbrueckii, Lactobacillus delbrueckii subsp. bulgaricus,Lactobacillus delbrueckii subsp. delbrueckii, Lactobacillus delbrueckiisubsp. lactis, Lactobacillus divergens, Lactobacillus farciminis,Lactobacillus fermentum, Lactobacillus fornicalis, Lactobacillusfructivorans, Lactobacillus fructosus, Lactobacillus gallinarum,Lactobacillus gasseri, Lactobacillus graminis, Lactobacillushalotolerans, Lactobacillus hamsteri, Lactobacillus helveticus,Lactobacillus heterohiochii, Lactobacillus hilgardii, Lactobacillushomohiochii, Lactobacillus iners, Lactobacillus intestinalis,Lactobacillus jensenii, Lactobacillus johnsonii, Lactobacillus kandleri,Lactobacillus kefiri, Lactobacillus kefiranofaciens, Lactobacilluskefirgranum, Lactobacillus kunkeei, Lactobacillus lactis, Lactobacillusleichmannii, Lactobacillus lindneri, Lactobacillus malefermentans,Lactobacillus mali, Lactobacillus maltaromicus, Lactobacillusmanihotivorans, Lactobacillus minor, Lactobacillus minutus,Lactobacillus mucosae, Lactobacillus murinus, Lactobacillus nagelii,Lactobacillus oris, Lactobacillus panis, Lactobacillus parabuchneri,Lactobacillus paracasei, Lactobacillus paracasei subsp. paracasei,Lactobacillus paracasei subsp. tolerans, Lactobacillus parakefiri,Lactobacillus paralimentarius, Lactobacillus paraplantarum,Lactobacillus pentosus, Lactobacillus perolens, Lactobacillus piscicola,Lactobacillus plantarum, Lactobacillus pontis, Lactobacillus reuteri,Lactobacillus rhamnosus, Lactobacillus rimae, Lactobacillus rogosae,Lactobacillus ruminis, Lactobacillus sakei, Lactobacillus sakei subsp.camosus, Lactobacillus sakei subsp. sakei, Lactobacillus salivarius,Lactobacillus salivarius subsp. salicinius, Lactobacillus salivariussubsp. salivarius, Lactobacillus sanfranciscensis, Lactobacillussharpeae, Lactobacillus suebicus, Lactobacillus trichodes, Lactobacillusuli, Lactobacillus vaccinostercus, Lactobacillus vaginalis,Lactobacillus viridescens, Lactobacillus vitulinus, Lactobacillusxylosus, Lactobacillus yamanashiensis, Lactobacillus yamanashiensissubsp. mali, Lactobacillus yamanashiensis subsp. Yamanashiensis,Lactobacillus zeae, Bifidobacterium adolescentis, Bifidobacteriumangulatum, Bifidobacterium bifidum, Bifidobacterium breve,Bifidobacterium catenulatum, Bifidobacterium longum, and Bifidobacteriuminfantis. In some examples, the LAB is Lactococcus lactis (LL).

In further examples, the bacterium is selected from the group consistingof Enterococcus alcedinis, Enterococcus aquimarinus, Enterococcus asini,Enterococcus avium, Enterococcus caccae, Enterococcus camelliae,Enterococcus canintestini, Enterococcus canis, Enterococcuscasseliflavus, Enterococcus cecorum, Enterococcus columbae, Enterococcusdevriesei, Enterococcus diestrammenae, Enterococcus dispar, Enterococcusdurans, Enterococcus eurekensis, Enterococcus faecalis, Enterococcusfaecium, Enterococcus gallinarum, Enterococcus gilvus, Enterococcushaemoperoxidus, Enterococcus hermanniensis, Enterococcus hirae,Enterococcus italicus, Enterococcus lactis, Enterococcus lemanii,Enterococcus malodoratus, Enterococcus moraviensis, Enterococcusmundtii, Enterococcus olivae, Enterococcus pallens, Enterococcusphoeniculicola, Enterococcus plantarum, Enterococcus pseudoavium,Enterococcus quebecensis, Enterococcus raffinosus, Enterococcus ratti,Enterococcus rivorum, Enterococcus rotai, Enterococcus saccharolyticus,Enterococcus silesiacus, Enterococcus solitarius, Enterococcussulfureus, Enterococcus termitis, Enterococcus thailandicus,Enterococcus ureasiticus, Enterococcus ureilyticus, Enterococcusviikkiensis, Enterococcus villorum, and Enterococcus xiangfangensis,

In further examples, the bacterium is selected from the group consistingof Streptococcus agalactiae, Streptococcus anginosus, Streptococcusbovis, Streptococcus canis, Streptococcus constellatus, Streptococcusdysgalactiae, Streptococcus equinus, Streptococcus iniae, Streptococcusintermedius, Streptococcus milleri, Streptococcus mitis, Streptococcusmutans, Streptococcus oralis, Streptococcus parasanguinis, Streptococcusperoris, Streptococcus pneumoniae, Streptococcus pseudopneumoniae,Streptococcus pyogenes, Streptococcus ratti, Streptococcus salivarius,Streptococcus tigurinus, Streptococcus thermophilus, Streptococcussanguinis, Streptococcus sobrinus, Streptococcus suis, Streptococcusuberis, Streptococcus vestibularis, Streptococcus viridans, andStreptococcus zooepidemicus.

In a particular aspect of the present invention, the gram-positive foodgrade bacterial strain is Lactococcus lactis or any of its subspecies,including Lactococcus lactis subsp. Cremoris, Lactococcus lactis subsp.Hordniae, and Lactococcus lactis subsp. Lactis. In another aspect of thepresent invention, the recombinant gram-positive bacterial strains is abiologically contained system, such as the plasmid free Lactococcuslactis strain MG1363, that lost the ability of normal growth and acidproduction in milk (Gasson, M. J. (1983) J. Bacteriol. 154:1-9); or thethreonine- and pyrimidine-auxotroph derivative L. lactis strains(Sorensen et al. (2000) Appl. Environ. Microbiol. 66:1253-1258; Glentinget al. (2002) 68:5051-5056).

In one embodiment of the present invention, the recombinant bacterialhost-vector system is a biologically contained system. Biologicalcontainment is known to the person skilled in the art and can berealized by the introduction of an auxotrophic mutation, for example asuicidal auxotrophic mutation such as the ThyA mutation, or itsequivalents, debilitating DNA synthesis. Other examples of auxotrophicmutations can debilitate RNA, cell wall or protein synthesis.Alternatively, the biological containment can be realized at the levelof the plasmid carrying the gene encoding the IL-10 polypeptide or IL-10variant, such as, for example, by using an unstable episomal construct,which is lost after a few generations. Several levels of containment,such as plasmid instability and auxotrophy, can be combined to ensure ahigh level of containment, if desired.

Constructs

In the present invention, the microorganism (e.g., the non-pathogenicgram-positive bacterium) delivers the IL-10 polypeptide and theT1D-specific antigen (e.g., PINS) at the intended site, i.e., themucosa. For example, the microorganism (e.g., LAB) expresses the IL-10polypeptide, after which the IL-10 polypeptide is secreted (if asecreted form of IL-10 is used). Hence, in a particular embodiment themicroorganism (e.g., LAB), such as L. lactis, expresses IL-10 and PINSat the site of an intended mucosa, e.g., in the gastrointestinal tract.

Use of an operon enables expression of the IL-10 polypeptide andT1D-specific antigen polypeptide (e.g., PINS) to be coordinated.Polycistronic expression systems in bacterial host cells are described,e.g., in U.S. Patent Application No. 2014/0105863 to Vanden-Broucke etal., which is incorporated herein by reference in its entirety.

In an embodiment the present invention relates to stably transfectedmicroorganisms, i.e., microorganisms in which the gene coding for theIL-10 polypeptide and the T1D-specific antigen (e.g., PINS) gene hasbeen integrated into the host cell's genome. Techniques for establishingstably transfected microorganisms are known in the art. For instance,the IL-10 polypeptide and the T1D-specific antigen (e.g., PINS) gene maybe cloned into the host's genome, e.g. in the chromosome, via homologousrecombination. In some embodiments, an essential gene of the host isdisrupted by the homologous recombination event, such as deletion of thegene, one or more amino acid substitutions leading to an inactive formof the protein encoded by the essential gene, or to a frameshiftmutation resulting in a truncated form of the protein encoded by theessential gene. In an embodiment, the essential gene is the thyA gene. Apreferred technique is described, e.g., in WO 02/090551, which isincorporated herein by reference in its entirety. The plasmid may be aself-replicating, for example carrying one or more genes of interest andone or more resistance markers. Then, the transforming plasmid can beany plasmid, as long as it cannot complement the disrupted essentialgene, e.g., thyA gene. Alternatively, the plasmid is an integrativeplasmid. In the latter case, the integrative plasmid itself may be usedto disrupt the essential gene, by causing integration at the locus ofthe essential gene, e.g., thyA site, because of which the function ofthe essential gene, e.g., the thyA gene, is disrupted. In some cases,the essential gene, such as the thyA gene, is replaced by doublehomologous recombination by a cassette comprising the gene or genes ofinterest, flanked by targeting sequences that target the insertion tothe essential gene, such as the thyA target site. It will be appreciatedthat that these targeting sequences are sufficiently long andsufficiently homologous to enable integration of the gene of interestinto the target site. In some examples, an IL-10 expression cassette ofthe present disclosure is integrated at the thyA locus.

The genetic construct encoding the IL-10 polypeptide and theT1D-specific antigen (e.g., PINS) may be integrated into the microbialgenomic DNA, e.g., bacterial or yeast chromosome, e.g., Lactococcuschromosome. In the latter case, a single or multiple copies of thenucleic acid may be integrated; the integration may occur at a randomsite of the chromosome or, as described above, at a predetermined sitethereof, for example at a predetermined site, such as, in a non-limitingexample, in the thyA locus of Lactococcus, e.g., Lactococcus lactis.

Hence, in an embodiment, the genetic construct encoding the IL-10polypeptide and the T1D-specific antigen (e.g., PINS) may furthercomprise sequences configured to effect insertion of the geneticconstruct into the genome, e.g., a chromosome, of a host cell.

In some examples, insertion of the genetic construct into particularsites within a genome, e.g., chromosome, of a host cell may befacilitated by homologous recombination. For instance, the geneticconstruct the invention may comprise one or more regions of homology tothe said site of integration within the genome e.g., a chromosome, ofthe host cell. The sequence at the said genome, e.g., chromosome, sitemay be natural, i.e., as occurring in nature, or may be an exogenoussequence introduced by previous genetic engineering. For instance, theregion(s) of homology may be at least 50 bp, 100 bp, 200 bp, 300 bp, 400bp, 500 bp, 600 bp 700 bp, 800 bp, 900 bp, 1000 bp, or more.

In one example, two regions of homology may be included, one flankingeach side of the relevant expression units present in the geneticconstruct of the invention. Such configuration may advantageously insertthe relevant sequences, i.e., at least the ones encoding and effectingthe expression of the antigen of interest, in host cells. Ways ofperforming homologous recombination, especially in bacterial hosts, andselecting for recombinants, are generally known in the art.

Transformation methods of microorganisms are known to the person skilledin the art, such as for instance protoplast transformation andelectroporation.

A high degree of expression can be achieved by using homologousexpression and/or secretion signals on the expression vectors present inthe microorganism, e.g., L. lactis. Expression signals will be apparentto the person skilled in the art. The expression vector can be optimizedfor expression depending on the microorganism, e.g., L. lactis, it isincorporated in. For instance, specific expression vectors that gavesufficient levels of expression in Lactococcus, Lactobacillus lactis,casei and plantarum are known. Moreover, systems are known which havebeen developed for the expression of heterologous antigens in thenon-pathogenic, non-colonizing, non-invasive food-grade bacteriumLactococcus lactis (see UK patent GB2278358B, which is incorporatedherein by reference). A particularly preferred construct according tothe invention comprises the multi-copy expression vector described inPCT/NL95/00135 (WO-A-96/32487), in which the nucleotide sequenceencoding the IL-10 polypeptide and/or the T1D-specific antigen (e.g.,PINS) has been incorporated. Such a construct is particularly suitablefor expression of a desired antigen in a lactic acid bacterium, inparticular in a Lactobacillus, at a high level of expression, and alsocan be used advantageously to direct the expressed product to thesurface of the bacterial cell. The constructs (e.g., of PCT/NL95/00135)may be characterized in that the nucleic acid sequence encoding theIL-10 polypeptide and/or T1D-specific antigen (e.g., PINS) is precededby a 5′ non-translated nucleic acid sequence comprising at least theminimal sequence required for ribosome recognition and RNAstabilization. This can be followed by a translation initiation codonwhich may be (immediately) followed by a fragment of at least 5 codonsof the 5′ terminal part of the translated nucleic acid sequence of agene of a lactic acid bacterium or a structural or functional equivalentof the fragment. The fragment may also be controlled by the promoter.The contents of PCT/NL95/00135, including the differing embodimentsdisclosed therein, and all other documents mentioned in thisspecification, are incorporated herein by reference. One aspect of thepresent invention provides a method which permits the high levelregulated expression of heterologous genes in the host and the couplingof expression to secretion. In another embodiment, the T7 bacteriophageRNA polymerase and its cognate promoter are used to develop a powerfulexpression system according to WO 93/17117, which is incorporated hereinby reference. In one embodiment, the expression plasmid is derived frompT1 NX (GenBank: HM585371.1).

A promoter employed in accordance with the present invention is in somecases expressed constitutively in the bacterium. The use of aconstitutive promoter avoids the need to supply an inducer or otherregulatory signal for expression to take place. In some cases, thepromoter directs expression at a level at which the bacterial host cellremains viable, i.e., retains some metabolic activity, even if growth isnot maintained. Advantageously then, such expression may be at a lowlevel. For example, where the expression product accumulatesintracellularly, the level of expression may lead to accumulation of theexpression product at less than about 10% of cellular protein, about orless than about 5%, for example about 1-3%. The promoter may behomologous to the bacterium employed, i.e., one found in that bacteriumin nature. For example, a Lactococcal promoter may be used in aLactococcus. A preferred promoter for use in Lactococcus lactis (orother Lactococci) is “P1” derived from the chromosome of Lactococcuslactis (Waterfield, N R, Lepage, R W F, Wilson, P W, et al. (1995). “Theisolation of lactococcal promoters and their use in investigatingbacterial luciferase synthesis in Lactococcus lactis” Gene 165(1):9-15).Other examples of a promoter include, the usp45 promoter, the gapBpromoter, and the hllA promoter. Other useful promoters are described inU.S. Pat. No. 8,759,088 to Steidler et al., and in U.S. PatentApplication No. 2014/0105863 to Vanden-Broucke et al., the disclosuresof which are incorporated herein by reference in their entirety. In someexamples, a nucleic acid encoding an IL-10 polypeptide is placed underan hllA promoter.

The nucleic acid construct or constructs may comprise a nucleic acidencoding a secretory signal sequence. Thus, in some embodiments thenucleic acid encoding IL-10 and/or the T1D-specific antigen (e.g., PINS)may provide for secretion of the polypeptides, e.g., by appropriatelycoupling a nucleic acid sequence encoding a signal sequence to thenucleic acid sequence encoding the polypeptide). Ability of a bacteriumharboring the nucleic acid to secrete the antigen may be tested in vitroin culture conditions which maintain viability of the organism.Preferred secretory signal sequences include any of those with activityin Gram positive organisms such as Bacillus, Clostridium andLactobacillus. Such sequences may include the α-amylase secretion leaderof Bacillus amyloliquetaciens or the secretion leader of theStaphylokinase enzyme secreted by some strains of Staphylococcus, whichis known to function in both Gram-positive and Gram-negative hosts (see“Gene Expression Using Bacillus,” Rapoport (1990) Current Opinion inBiotechnology 1:21-27), or leader sequences from numerous other Bacillusenzymes or S-layer proteins (see pp 341-344 of Harwood and Cutting,“Molecular Biological Methods for Bacillus,” John Wiley & Co. 1990). Inone embodiment, said secretion signal is derived from usp45 (VanAsseldonk et al. (1993) Mol. Gen. Genet. 240:428-434). Such secretionleader is referred to herein, e.g., as SSusp45. In some embodiments, theIL-10 polypeptide is constitutively secreted using SSusp45. In otherexamples, the PINS polypeptide is secreted using SSusp45. In yet otherexamples, both the IL-10 polypeptide and the PINS polypeptide aresecreted using SSusp45.

A person of ordinary skill in the art will appreciate that the optimalamount of IL-10 and PINS to be delivered to the subject using themethods of the present disclosure varies, e.g., with the microorganismexpressing the IL-10 polypeptide and the PINS polypeptide, and thegenetic constructs, e.g., the strength of the promoter used in thegenetic constructs. Typically, the microorganism will be administered inan amount equivalent to a particular amount of expressed IL-10polypeptide and PINS polypeptide, or in an amount which generates adesired PK profile for the respective IL-10 polypeptide or PINSpolypeptide in the respective subject. Exemplary daily IL-10 polypeptideor PINS polypeptide doses are from about 10 fg to about 100 μg of activepolypeptide per day. Other exemplary dose ranges are from about 1 pg toabout 100 μg per day; or from about 1 ng to about 100 μg per day.

The above doses may be realized by administering to the subjecteffective amounts of the microorganism per day, wherein themicroorganism is adapted to express a sufficient amount of IL-10 and aT1D-specific antigen (e.g., PINS) to realize the desired dose, such asthose above. The microorganism secreting the IL-10 polypeptide and theT1D-specific antigen (e.g., PINS polypeptide) may be delivered in a doseof from about 10⁴ colony forming units (cfu) to about 10¹² cfu per day,in particular from about 10⁶ cfu to about 10¹² cfu per day, more inparticular from about 10⁹ cfu to about 10¹² cfu per day. The amount ofsecreted IL-10 and T1D-specific antigen (e.g., PINS polypeptide) can bedetermined based on cfu, for example in accordance with the methodsdescribed in Steidler et al., Science 2000; 289(5483):1352-1355, or byusing ELISA. For example, a particular microorganism may secrete atleast about 1 ng to about 1 μg IL-10 per 10⁹ cfu. Based thereon, theskilled person can calculate the range of IL-10 polypeptide secreted atother cfu doses.

Each of the above doses/dose ranges may be administered in connectionwith any dosing regimen as described herein. The daily dose may beadministered in 1, 2, 3, 4, 5, or 6 portions throughout the day. Furtherthe daily doses may be administered for any number of days, with anynumber of rest periods between administration periods. For example, thesubject may be administered the microorganism at a dose equivalent toabout 0.01 to about 3 M IU of IL-10/day or every other day, for a periodof at least about 1 week, at least about 2 weeks, at least about 3weeks, at least about 4 weeks, at least about 5 weeks, or at least about6 weeks. In some examples, the subject is administered the microorganismat a dose equivalent to about 0.1 to about 5 MIU/day, or about 0.3 toabout 3 MIU, e.g., for about 5 days, about 7 days, or about 14 days.Exemplary doses are described, e.g., in Hartemann et al., LancetDiabetes Endocrinol. 2013, 1(4): 295-305, the disclosure of which isincorporated herein by reference in its entirety.

Formulations and Regimens

In some methods of the present disclosure, the IL-10 polypeptide and theT1D-specific antigen (e.g., PINS) are administered (delivered) to asubject (e.g., a human T1D-patient) using a microorganism (e.g., LAB)producing both the IL-10 polypeptide and the T1D-specific antigen (e.g.,PINS) polypeptide.

In some embodiments, the microorganism (e.g., LAB), optionally containedin a composition (e.g., a pharmaceutical composition) of the presentdisclosure or a unit dosage form of the present disclosure, will beadministered, once, twice, three, four, five, or six times daily, e.g.,using an oral formulation. In some embodiments, the microorganism isadministered every day, every other day, once per week, twice per week,three times per week, or four times per week. In other embodiments,treatment occurs once every two weeks. In other embodiments, treatmentoccurs once every three weeks. In other embodiments, treatment occursonce per month.

The duration of a treatment cycle is, for example, 7 days to thesubject's lifetime, as needed to treat or reverse T1D, or preventrelapse. In some embodiments, a treatment cycle lasts for about 30 daysto about 2 years. In other embodiments, the subject will have atreatment cycle that lasts from 30 days to 1.5 years. In otherembodiments, the subject will have a treatment cycle that lasts from 30days to 1 year. In other embodiments, the subject will have a treatmentcycle that lasts from 30 days to 11 months. In other embodiments, thesubject will have a treatment cycle that lasts from 30 days to 10months. In other embodiments, the subject will have a treatment cyclethat lasts from 30 days to 9 months. In other embodiments, the subjectwill have a treatment cycle that lasts from 30 days to 8 months. Inother embodiments, the subject will have a treatment cycle that lastsfrom 30 days to 7 months. In other embodiments, the subject will have atreatment cycle that lasts from 30 days to 6 months. In otherembodiments, the subject will have a treatment cycle that lasts from 30days to 5 months. In other embodiments, the subject will have atreatment cycle that lasts from 30 days to 4 months. In otherembodiments, the subject will have a treatment cycle that lasts from 30days to 3 months. In other embodiments, the subject will have atreatment cycle that lasts from 30 days to 2 months.

In further embodiments, the treatment cycle will be based on the levelof markers that track the progress of disease, including glycemic index,insulin autoantibody (IAA) levels, fragment C levels, and insulitis.Preferably, the subject is at early onset diabetes. A subject may alsobe assessed for the level of Treg cells that suppress the immuneresponse to beta cells. In an exemplary embodiment, the subject istreated at least until there is no further disease progression,preferably until there is a return of normoglycemia. The patient may betreated for an additional period to ensure a population of Treg cellsthat suppress and reverse disease. A subject may also be monitored andtreated at the first appearance of any indicia of reemergent disease.

Daily maintenance doses can be given for a period clinically desirablein the subject, for example from 1 day up to several years (e.g. for thesubject's entire remaining life); for example from about (2, 3 or 5days, 1 or 2 weeks, or 1 month) upwards and/or for example up to about(5 years, 1 year, 6 months, 1 month, 1 week, or 3 or 5 days).Administration of the daily maintenance dose for about 3 to about 5 daysor for about 1 week to about 1 year is typical. Nevertheless, unit dosesshould for example be administered from twice daily to once every twoweeks until a therapeutic effect is observed.

The microorganisms producing the IL-10 polypeptide and the T1D-specificantigen (e.g., PINS) polypeptide may be administered to the subject inmono- or combination therapy (e.g., using a co-therapeutic regimen) forthe treatment of T1D. “The term “co-therapy,” “co-therapeutic” orvariation thereof refers to a treatment regimen, in which the subject isadministered at least one additional therapeutically active agent, suchas an additional immuno-modulating compound. Thus, in some embodiments,the compositions of the present disclosure include additionaltherapeutically active agents. In some embodiments, the compositions ofthe present disclosure contain at least one additional immuno-modulatingsubstance, such as antibodies (e.g., anti-CD3 antibodies). In someexamples, the methods of the present disclosure further includeadministering to the subject (e.g., a human patient) an additionalimmuno-modulating substance, such as antibodies (e.g., anti-CD3antibodies).

Pharmaceutical Compositions and Carriers

The microorganism (e.g., bacteria, such as LAB described herein) may beadministered in pure form, combined with other active ingredients,and/or combined with pharmaceutically acceptable (i.e., nontoxic)excipients or carriers. The term “pharmaceutically acceptable” is usedherein in accordance with its art-recognized meaning and refers tocarriers that are compatible with the other ingredients of apharmaceutical composition, and are not deleterious to the recipientthereof.

The compositions of the present disclosure can be prepared in any knownor otherwise effective dosage or product form suitable for delivery ofthe microorganism (e.g., bacteria) to the mucosa, which would includepharmaceutical compositions and dosage forms as well as nutritionalproduct forms.

In some embodiments, the pharmaceutical composition (i.e., formulation)is an oral pharmaceutical composition. In some examples according tothis embodiment, the formulation or pharmaceutical composition comprisesthe non-pathogenic microorganism in a dried form (e.g., dry-powder form;e.g., freeze-dried form) or in compacted form thereof, optionally incombination with other dry carriers. Oral formulations will generallyinclude an inert diluent carrier or an edible carrier.

In some examples, the oral formulation comprises a coating or utilizesan encapsulation strategy, which facilitates the delivery of theformulation into the intestinal tract, and/or allows the microorganismbe released and hydrated in the intestinal tract (e.g., the ileum, smallintestine, or the colon). Once the microorganism is released from theformulation and sufficiently hydrated, it begins expressing thebioactive polypeptides, which are subsequently released into thesurroundings, or expressed on the surface of the microorganism. Suchcoating and encapsulation strategies (i.e., delayed-release strategies)are known to those of skill in the art. See, e.g., U.S. Pat. No.5,972,685; WO 2000/18377; and WO 2000/22909, the disclosures of whichare incorporated herein by reference in their entirety.

In some embodiments, the disclosure provides a pharmaceuticalcomposition comprising the microorganism (e.g., the non-pathogenicbacteria) in a lyophilized or freeze dried form, optionally inconjunction with other components, such as dextrans, sodium glutamate,and polyols. Exemplary freeze dried compositions are described, e.g., inU.S. Patent Application No. 2012/0039853 to Corveleyn et al., thedisclosure of which is incorporated herein by reference in its entirety.Exemplary formulations comprise freeze-dried bacteria (e.g., atherapeutically effective amount of the bacteria) and a pharmaceuticallyacceptable carrier. Freeze-dried bacteria may be prepared in the form ofcapsules, tablets, granulates and powders, each of which may beadministered orally. Alternatively, freeze-dried bacteria may beprepared as aqueous suspensions in suitable media, or lyophilizedbacteria may be suspended in a suitable medium, such as a drink, justprior to use. Such composition may additionally contain a stabilizingagent useful to maintain a stable suspension, e.g., withoutprecipitation, aggregation, or floating of the bacterial biomass.

For oral administration, the formulation may be a gastro-resistant oraldosage form. For example, the oral dosage form (e.g., capsules, tablets,pellets, micro-pellets, granulates, and the like) may be coated with athin layer of excipient (usually polymers, cellulosic derivatives and/orlipophilic materials) that resists dissolution or disruption in thestomach, but not in the intestine, thereby allowing transit through thestomach in favor of disintegration, dissolution and absorption in theintestine (e.g., the small intestine, or the colon).

In some examples, oral formulations may include compounds providingcontrolled release, sustained release, or prolonged release of themicroorganism, and thereby provide controlled release of the desiredprotein encoded therein. These dosage forms (e.g., tablets or capsules)typically contain conventional and well known excipients, such aslipophilic, polymeric, cellulosic, insoluble, and/or swellableexcipients. Controlled release formulations may also be used for anyother delivery sites including intestinal, colon, bioadhesion orsublingual delivery (i.e., dental mucosal delivery) and bronchialdelivery. When the compositions of the invention are to be administeredrectally or vaginally, pharmaceutical formulations may includesuppositories and creams. In this instance, the host cells are suspendedin a mixture of common excipients also including lipids. Each of theaforementioned formulations are well known in the art and are described,for example, in the following references: Hansel et al. (1990,Pharmaceutical dosage forms and drug delivery systems, 5th edition,William and Wilkins); Chien 1992, Novel drug delivery system, 2ndedition, M. Dekker); Prescott et al. (1989, Novel drug delivery, J.Wiley & Sons); Gazzaniga et al., (1994, Oral delayed release system forcolonic specific delivery, Int. J. Pharm. 108:77-83).

In some embodiments, the oral formulation includes compounds that canenhance mucosal delivery and/or mucosal uptake of the bioactivepolypeptides expressed by the microorganism. In other examples, theformulation includes compounds, which enhance the viability of themicroorganism within the formulation, and/or once released.

The bacteria of the invention can be suspended in a pharmaceuticalformulation for administration to the human or animal having the diseaseto be treated. Such pharmaceutical formulations include but are notlimited to live gram-positive bacteria and a medium suitable foradministration. The bacteria may be lyophilized in the presence ofcommon excipients such as lactose, other sugars, alkaline and/or alkaliearth stearate, carbonate and/or sulphate (e.g., magnesium stearate,sodium carbonate and sodium sulphate), kaolin, silica, flavorants andaromas. Bacteria so-lyophilized may be prepared in the form of capsules,tablets, granulates and powders (e.g., a mouth rinse powder), each ofwhich may be administered by the oral route. Alternatively, somegram-positive bacteria may be prepared as aqueous suspensions insuitable media, or lyophilized bacteria may be suspended in a suitablemedium just prior to use, such medium including the excipients referredto herein and other excipients such as glucose, glycine and sodiumsaccharinate.

In some examples, the microorganism is locally delivered to thegastrointestinal tract of the subject using any suitable method. Forexample, microsphere delivery systems could be employed to enhancedelivery to the gut. Microsphere delivery systems include microparticleshaving a coating that provides localized release into thegastrointestinal tract of the subject (e.g., controlled releaseformulations such as enteric-coated formulations and colonicformulations).

For oral administration, gastroresistant oral dosage forms may beformulated, which dosage forms may also include compounds providingcontrolled release of the gram-positive bacteria and thereby providecontrolled release of the desired protein encoded therein (e.g., IL-10).For example, the oral dosage form (including capsules, tablets, pellets,granulates, powders) may be coated with a thin layer of excipient (e.g.,polymers, cellulosic derivatives and/or lipophilic materials) thatresists dissolution or disruption in the stomach, but not in theintestine, thereby allowing transit through the stomach in favor ofdisintegration, dissolution and absorption in the intestine.

The oral dosage form may be designed to allow slow release of thegram-positive bacteria and of the produced exogenous proteins, forinstance as controlled release, sustained release, prolonged release,sustained action tablets or capsules. These dosage forms usually containconventional and well-known excipients, such as lipophilic, polymeric,cellulosic, insoluble, and/or swellable excipients. Such formulationsare well-known in the art and are described, for example, in thefollowing references: Hansel et al., Pharmaceutical dosage forms anddrug delivery systems, 5th edition, William and Wilkins, 1990; Chien1992, Novel drug delivery system, 2nd edition, M. Dekker; Prescott etal., Novel drug delivery, J. Wiley & Sons, 1989; and Gazzaniga et al.,Int. J. Pharm. 108: 77-83 (1994).

The pharmaceutical dosage form (e.g. capsule) may be coated withpH-dependent Eudragit polymers to obtain gastric juice resistance andfor the intended delivery at the terminal ileum and colon, where thepolymers dissolve at pH 6.5. By using other Eudragit polymers or adifferent ratio between the polymers, the delayed release profile couldbe adjusted, to release the bacteria for example in the duodenum orjejenum.

Pharmaceutical compositions contain at least one pharmaceuticallyacceptable carrier. Non-limiting examples of suitable excipients,diluents, and carriers include preservatives, inorganic salts, acids,bases, buffers, nutrients, vitamins, fillers and extenders such asstarch, sugars, mannitol, and silicic derivatives; binding agents suchas carboxymethyl cellulose and other cellulose derivatives, alginates,gelatin, and polyvinyl pyrolidone; moisturizing agents such asglycerol/disintegrating agents such as calcium carbonate and sodiumbicarbonate; agents for retarding dissolution such as paraffin;resorption accelerators such as quaternary ammonium compounds; surfaceactive agents such as acetyl alcohol, glycerol monostearate; adsorptivecarriers such as kaolin and bentonite; carriers such as propylene glycoland ethyl alcohol, and lubricants such as talc, calcium and magnesiumstearate, and solid polyethyl glycols.

Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. Tablets, pills, capsules,troches and the like can contain any of the following ingredients, orcompounds of a similar nature: a binder such as microcrystallinecellulose, gum tragacanth or gelatin; an excipient such as starch orlactose, a dispersing agent such as alginic acid, Primogel, or cornstarch; a lubricant such as magnesium stearate; a glidant such ascolloidal silicon dioxide; a sweetening agent such as sucrose orsaccharin; or a flavoring agent such as peppermint, methyl salicylate,or orange flavoring. When the dosage unit form is a capsule, it cancontain, in addition to material of the above type, a liquid carriersuch as a fatty oil. In addition, dosage unit forms can contain variousother materials that modify the physical form of the dosage unit, forexample, coatings of sugar, shellac, or enteric agents. Further, a syrupmay contain, in addition to the active compounds, sucrose as asweetening agent and certain preservatives, dyes, colorings, andflavorings. It will be appreciated that the form and character of thepharmaceutically acceptable carrier is dictated by the amount of activeingredient with which it is to be combined, the route of administrationand other well-known variables. The carrier(s) must be “acceptable” inthe sense of being compatible with the other ingredients of theformulation and not deleterious to the recipient thereof.

Alternative preparations for administration include sterile aqueous ornonaqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are dimethylsulfoxide, alcohols, propylene glycol,polyethylene glycol, vegetable oils such as olive oil and injectableorganic esters such as ethyl oleate. Aqueous carriers include mixturesof alcohols and water, buffered media, and saline. Intravenous vehiclesinclude fluid and nutrient replenishers, electrolyte replenishers, suchas those based on Ringer's dextrose, and the like. Preservatives andother additives may also be present such as, for example,antimicrobials, anti-oxidants, chelating agents, inert gases, and thelike. Various liquid formulations are possible for these deliverymethods, including saline, alcohol, DMSO, and water based solutions.

Oral aqueous formulations include excipients, such as pharmaceuticalgrades of mannitol, lactose, starch, magnesium stearate, sodiumsaccharine, cellulose, magnesium carbonate and/or the like. Thesecompositions take the form of solutions such as mouthwashes andmouthrinses, further comprising an aqueous carrier such as for examplewater, alcoholic/aqueous solutions, saline solutions, parenteralvehicles such as sodium chloride, Ringer's dextrose, and the like.

Aqueous mouthwash formulations are well-known to those skilled in theart. Formulations pertaining to mouthwashes and oral rinses arediscussed in detail, for example, in U.S. Pat. Nos. 6,387,352,6,348,187, 6,171,611, 6,165,494, 6,117,417, 5,993,785, 5,695,746,5,470,561, 4,919,918, U.S. Patent Appl. No. 2004/0076590, U.S. PatentAppl. No. 2003/0152530, and U.S. Patent Appl. No. 2002/0044910, each ofwhich is herein specifically incorporated by reference into this sectionof the specification and all other sections of the specification.

Other additives may be present in the formulations of the presentdisclosure, such as flavoring, sweetening or coloring agents, orpreservatives. Mint, such as from peppermint or spearmint, cinnamon,eucalyptus, citrus, cassia, anise and menthol are examples of suitableflavoring agents. Flavoring agents are for example present in the oralcompositions in an amount in the range of from 0 to 3%; up to 2%, suchas up to 0.5%, e.g., around 0.2%, in the case of liquid compositions.

Sweeteners include artificial or natural sweetening agents, such assodium saccharin, sucrose, glucose, saccharin, dextrose, levulose,lactose, mannitol, sorbitol, fructose, maltose, xylitol, thaumatin,aspartame, D-tryptophan, dihydrochalcones, acesulfame, and anycombination thereof, which may be present in an amount in the range offrom 0 to 2%, for example, up to 1% w/w, such as 0.05 to 0.3% w/w of theoral composition.

Coloring agents are suitable natural or synthetic colors, such astitanium dioxide or CI 42090, or mixtures thereof. Coloring agents arepreferably present in the compositions in an amount in the range of from0 to 3%; for example, up to 0.1%, such as up to 0.05%, e.g., around0.005-0.0005%, in the case of liquid compositions. Of the usualpreservatives, sodium benzoate is preferred in concentrationsinsufficient substantially to alter the pH of the composition, otherwisethe amount of buffering agent may need to be adjusted to arrive at thedesired pH.

Other optional ingredients include humectants, surfactants (non-ionic,cationic or amphoteric), thickeners, gums and binding agents. Ahumectant adds body to the formulation and retains moisture in adentifrice composition. In addition, a humectant helps to preventmicrobial deterioration during storage of the formulation. It alsoassists in maintaining phase stability and provides a way to formulate atransparent or translucent dentifrice.

Suitable humectants include glycerine, xylitol, glycerol and glycolssuch as propylene glycol, which may be present, for example, in anamount of up to 50% w/w each, but total humectant is in some cases notmore than about 60-80% w/w of the composition. For example, liquidcompositions may comprise up to about 30% glycerine plus up to about 5%,for example, about 2% w/w xylitol. Surfactants are preferably notanionic and may include polysorbate 20 or cocoamidobetaine or the likein an amount up to about 6%, for example, about 1.5 to 3%, w/w of thecomposition.

When the oral compositions of the invention are in a liquid form, it ispreferred to include a film-forming agent up to about 3% w/w of the oralcomposition, such as in the range of from 0 to 0.1%, for example, about0.001 to 0.01%, such as about 0.005% w/w of the oral composition.Suitable film-formers include (in addition to sodium hyaluronate) thosesold under the tradename Gantrez.

Liquid nutritional formulations for oral or enteral administration maycomprise one or more nutrients such as fats, carbohydrates, proteins,vitamins, and minerals. Many different sources and types ofcarbohydrates, lipids, proteins, minerals and vitamins are known and canbe used in the nutritional liquid embodiments of the present invention,provided that such nutrients are compatible with the added ingredientsin the selected formulation, are safe and effective for their intendeduse, and do not otherwise unduly impair product performance.

These nutritional liquids are for example formulated with sufficientviscosity, flow, or other physical or chemical characteristics toprovide a more effective and soothing coating of the mucosa whiledrinking or administering the nutritional liquid. These nutritionalembodiments also in some cases represent a balanced nutritional sourcesuitable for meeting the sole, primary, or supplemental nutrition needsof the individual.

Non-limiting examples of suitable nutritional liquids are described inU.S. Pat. No. 5,700,782 (Hwang et al.); U.S. Pat. No. 5,869,118 (Morriset al.); and U.S. Pat. No. 5,223,285 (DeMichele et al.), whichdescriptions are incorporated herein by reference in their entireties.

Nutritional proteins suitable for use herein can be hydrolyzed,partially hydrolyzed or non-hydrolyzed, and can be derived from anyknown or otherwise suitable source such as milk (e.g., casein, whey),animal (e.g., meat, fish), cereal (e.g., rice, corn), vegetable (e.g.,soy), or any combination thereof.

Fats or lipids suitable for use in the nutritional liquids include, butare not limited to, coconut oil, soy oil, corn oil, olive oil, saffloweroil, high oleic safflower oil, MCT oil (medium chain triglycerides),sunflower oil, high oleic sunflower oil, structured triglycerides, palmand palm kernel oils, palm olein, canola oil, marine oils, cottonseedoils, and any combination thereof. Carbohydrates suitable for use in thenutritional liquids may be simple or complex, lactose-containing orlactose-free, or any combination thereof. Non-limiting examples ofsuitable carbohydrates include hydrolyzed corn starch, maltodextrin,glucose polymers, sucrose, corn syrup, corn syrup solids, rice-derivedcarbohydrate, glucose, fructose, lactose, high fructose corn syrup andindigestible oligosaccharides such as fructo-oligosaccharides (FOS), andany combination thereof.

The nutritional liquids may further comprise any of a variety ofvitamins, non-limiting examples of which include vitamin A, vitamin D,vitamin E, vitamin K, thiamine, riboflavin, pyridoxine, vitamin B12,niacin, folic acid, pantothenic acid, biotin, vitamin C, choline,inositol, salts and derivatives thereof, and any combination thereof.

The nutritional liquids may further comprise any of a variety ofminerals known or otherwise suitable for us in patients at risk of orsuffering from T1D, non-limiting examples of which include calcium,phosphorus, magnesium iron, selenium, manganese, copper, iodine, sodium,potassium, chloride, and any combination thereof.

The microorganisms and in particular the yeast and bacteria of thepresent invention can also be formulated as elixirs or solutions forconvenient oral or rectal administration or as solutions appropriate forparenteral administration, for instance by intramuscular, subcutaneousor intravenous routes. Additionally, the nucleoside derivatives are alsowell suited for formulation as a sustained or prolonged release dosageforms, including dosage forms that release active ingredient only or insome cases in a particular part of the intestinal tract, for exampleover an extended or prolonged period of time to further enhanceeffectiveness. The coatings, envelopes, and protective matrices in suchdosage forms may be made, for example, from polymeric substances orwaxes well known in the pharmaceutical arts.

The compositions of the present invention include pharmaceutical dosageforms such as lozenges, troches or pastilles. These are typicallydiscoid-shaped solids containing the active ingredient in a suitablyflavored base. The base may be a hard sugar candy, glycerinated gelatin,or the combination of sugar with sufficient mucilage to give it form.Troches are placed in the mouth where they slowly dissolve, liberatingthe active ingredient for direct contact with the mucosa.

The troche embodiments of the present invention can be prepared, forexample, by adding water slowly to a mixture of the powdered active,powdered sugar, and a gum until a pliable mass is formed. A 7% acaciapowder can be used to provide sufficient adhesiveness to the mass. Themass is rolled out and the troche pieces cut from the flattened mass, orthe mass can be rolled into a cylinder and divided. Each cut or dividedpiece is shaped and allowed to dry, to thus form the troche dosage form.

If the active ingredient is heat labile, it may be made into a lozengepreparation by compression. For example, the granulation step in thepreparation is performed in a manner similar to that used for anycompressed tablet. The lozenge is made using heavy compression equipmentto give a tablet that is harder than usual as it is desirable for thedosage form to dissolve or disintegrate slowly in the mouth. Ingredientsare in some cases selected to promote slow-dissolving characteristics.

In a particular formulation of the present invention, the microorganismswill be incorporated in a bioadhesive carrier containing pregelatinizedstarch and cross-linked poly (acrylic acid) to form a bioadhesive tabletand a bioadhesive gel suitable for buccal application (i.e., havingprolonged bioadhesion and sustained drug delivery.

In an alternative embodiment, a powder mixture of non-pathogenic andnon-invasive bacterium according to the invention, bioadhesive polymers(pregelatinized starch and cross-linked poly (acrylic acid) coprocessedvia spray drying), sodium stearyl fumarate (lubricant), and siliciumdioxide (glidant) is processed into tablets (weight: 100 mg; diameter: 7mm). The methods for the production of these tablets are well known tothe person skilled in the art and has been described before for thesuccessful development of bioadhesive tablets containing various drugs(miconazol, testosterone, fluoride, ciprofloxacin) (Bruschi M. L. and deFreitas O., Drug Development and Industrial Pharmacy, 2005 31:293-310).All excipient materials are commercially available in pharmaceuticalgrades.

To optimize a formulation, the drug load in the tablets and the ratiobetween starch and poly (acrylic acid) will be varied. Based on previousresearch, the maximum drug load in the coprocessed bioadhesive carrieris about 60% (w/w) and the starch/poly (acrylic acid) ratio can bevaried between 75/25 and 95/5 (w/w). During the optimization study, thebioadhesive properties of the tablets and the drug release from thetablets are the main evaluation parameters, with the standard tabletproperties (hardness, friability) as secondary evaluation criteria.

The bacteria are incorporated into an aqueous dispersion ofpregelatinized starch and cross-linked poly (acrylic acid). This polymerdispersion is prepared via a standard procedure using a high shearmixer.

Similar to the tablet, the drug load of the gel and the starch/poly(acrylic acid) ratio need to be optimized in order to obtain a gelhaving optimal adherence to the esophageal mucosa. For a gel, theconcentration of the polymers in the dispersion is an additionalvariable as it determines the viscosity of the gel, hence itsmuco-adhesive properties.

The model to screen the bioadhesive properties of polymer dispersions tothe mucosa of esophagus has been described in detail by Batchelor et al.(Int. J. Pharm., 238:123-132, 2002).

Other routes and forms of administration include food preparationscontaining the live microorganisms. In some examples, the bioactivepolypeptide-expressing microorganism can be included into a dairyproduct.

The pharmaceutical compositions of the present invention can be preparedby any known or otherwise effective method for formulating ormanufacturing the selected dosage form. For example, the microorganismscan be formulated along with common, e.g., pharmaceutically acceptablecarriers, such as excipients and diluents, formed into oral tablets,capsules, sprays, lozenges, treated substrates (e.g., oral or topicalswabs, pads, or disposable, non-digestible substrate treated with thecompositions of the present invention); oral liquids (e.g., suspensions,solutions, emulsions), powders, suppositories, or any other suitabledosage form. In some embodiments, the present disclosure provides amethod for the manufacture of a pharmaceutical composition. Exemplarymethods include: contacting the microorganism (e.g., the non-pathogenicbacterium) containing the IL-10 gene and the T1D-specific antigen gene(or which is capable of expressing the IL-10 and the T1D-specificantigen) with a pharmaceutically acceptable carrier, thereby forming thepharmaceutical composition. In some examples, the method furtherincludes: growing the microorganism in a medium. The method may furtherinclude freeze-drying a liquid containing the microorganism, wherein theliquid optionally includes the pharmaceutically acceptable carrier.

Unit Dosage Forms

The current disclosure further provides unit dosage forms comprising acertain amount of a non-pathogenic microorganism optionally incombination with a food-grade or pharmaceutically acceptable carrier,wherein said non-pathogenic microorganism (e.g., the non-pathogenicgram-positive bacterium) comprises: an exogenous nucleic acid encodingan IL-10 polypeptide; and an exogenous nucleic acid encoding a type-1diabetes mellitus (T1D)-specific antigen (e.g., PINS). Exemplary unitdosage forms contain from about 1×10³ to about 1×10¹⁴ colony-formingunits (cfu) of the non-pathogenic microorganism (e.g., a non-pathogenicgram-positive bacterium). Other exemplary unit dosage forms contain fromabout 1×10⁴ to about 1×10¹³ colony-forming units (cfu) of anon-pathogenic microorganism (e.g., a non-pathogenic gram-positivebacterium), or from about 1×10⁴ to about 1×10¹² colony-forming units(cfu) of a non-pathogenic microorganism (e.g., a non-pathogenicgram-positive bacterium). In other embodiments, the unit dosage formcomprises from about 1×10⁵ to about 1×10¹² colony-forming units (cfu),or from about 1×10⁶ to about 1×10¹² colony-forming units (cfu) of thenon-pathogenic microorganism (e.g., the non-pathogenic gram-positivebacterium). In other embodiments, the unit dosage form comprises fromabout 1×10⁸ to about 1×10¹² colony-forming units (cfu), or from about1×10⁹ to about 1×10¹² colony-forming units (cfu) of the non-pathogenicmicroorganism (e.g., the non-pathogenic gram-positive bacterium). In yetother embodiments, the unit dosage form comprises from about 1×10⁹ toabout 1×10¹¹ colony-forming units (cfu), or from about 1×10⁹ to about1×10¹⁰ colony-forming units (cfu) of the non-pathogenic microorganism(e.g., the non-pathogenic gram-positive bacterium). In yet otherembodiments, the unit dosage form comprises from about 1×10⁷ to about1×10¹¹ colony-forming units (cfu), or from about 1×10⁸ to about 1×10¹⁰colony-forming units (cfu) of the non-pathogenic microorganism (e.g.,the non-pathogenic gram-positive bacterium).

In yet other embodiments, the unit dosage form comprises from about1×10⁹ to about 1×10¹⁰ colony-forming units (cfu), or from about 1×10⁹ toabout 100×10⁹ colony-forming units (cfu) of the non-pathogenicmicroorganism (e.g., the non-pathogenic gram-positive bacterium).

The unit dosage form can have any physical form or shape. In someembodiments, the unit dosage form is adapted for oral administration. Insome examples according to these embodiments, the unit dosage form is inthe form of a capsule, a tablet, or a granule. Exemplary capsulesinclude capsules filled with micro-granules. In some embodiments, thenon-pathogenic microorganism (e.g., the non-pathogenic gram-positivebacterium) contained in the dosage form is in a dry-powder form. Forexample, the microorganism is in a freeze-dried powder form, which isoptionally compacted and coated.

This compositions and methods can be better understood by reference tothe Examples that follow, but those skilled in the art will appreciatethat these are only illustrative of the invention as described morefully in the numbered embodiments and claims that follow. Additionally,throughout this application, various publications are cited. Thedisclosures of these publications are hereby incorporated by referencein their entirety.

EXAMPLES Example 1 Construction of a Clinical-Grade Lactococcus lactisSecreting hPINS and hIL10

A Lactococcus lactis strain (sAGX0407) secreting human PINS and humanIL10 was generated by replacement of the chromosomally-locatedthymidylate synthase (thyA) gene in an MG1363 parental strain by anexpression cassette for human PINS and IL10 using methods previouslydescribed. See, e.g., Steidler L. et al., Nat. Biotechnol. 2003;21:785-789; and Steidler L, Rottiers P; Annals of the New York Academyof Sciences 2006; 1072:176-186. Methods to introduce changes into the L.lactis chromosome make use of double homologous recombination. Aconditionally replicative carrier plasmid derived from pORI19 andcontaining an erythromycin selection marker, was constructed in therepA+L. lactis strain LL108. Carrier plasmids were designed in such waythat the cargo of interest was cloned in between up to 1 kb cross over(XO) areas, identical to the ones flanking the wild type sequence on thebacterial chromosome. This plasmid was introduced into MG1363 or any ofits derivatives (repA−). Resistant colonies were selected on agar platescontaining erythromycin and a first homologous recombination either atthe 5′ or 3′ target sites was verified by PCR screening. Release oferythromycin selection enabled the excision of the carrier plasmid fromthe bacterial chromosome by a second homologous recombination, at eitherthe 5′ or 3′ target site. The final genetic structure of theclinical-grade strain was extensively documented by both Sanger andIllumina full genome sequencing. There are no plasmids or residualerythromycin resistance in the final clinical strain. See, e.g.,Steidler, L., et al., Nat. Biotechnol. 2003, 21(7): 785-789.

sAGX0407 is a derivative of Lactococcus lactis (L. lactis) MG1363. InsAGX0407:

-   -   Thymidylate synthase gene (thyA; Gene ID: 4798358) is absent, to        warrant environmental containment (Steidler, L., et al., Nat.        Biotechnol. 2003, 21(7): 785-789).    -   At the position where thyA had been deleted, and downstream of        the constitutive promoter of the HU-like DNA-binding protein        gene (PhllA; Gene ID: 4797353), a gene encoding the secretion        leader of unidentified secreted 45-kDa protein gene (usp45; Gene        ID: 4797218; SSusp45) fused to the hil-10 gene, encoding human        interleukin-10 (hiL-10; UniProt: P22301, aa 19-178, variant P2A;        Steidler, L., et al., Nat. Biotechnol. 2003, 21(7): 785-789) is        inserted to allow expression and secretion of hIL-10.    -   Trehalose-6-phosphate phosphorylase gene (trePP; Gene        ID: 4797140) is absent, to allow accumulation of exogenously        added trehalose.    -   Trehalose-6-phosphate phosphatase gene (otsB; Gene ID: 1036914)        is positioned downstream of usp45 (Gene ID: 4797218) to        facilitate conversion of trehalose-6-phosphate to trehalose. The        otsB expression unit was transcriptionally and translationally        coupled to usp45 by use of the intergenic region (IR) preceding        the highly expressed L. lactis MG1363 50S ribosomal protein L30        gene (rpmD; Gene ID: 4797873).    -   The constitutive promoter of the HU-like DNA-binding protein        gene (PhllA; Gene ID: 4797353) is preceding the putative        phosphotransferase genes in the trehalose operon (trePTS;        llmg_0453 and llmg_0454; Gene ID: 4797778 and Gene ID: 4797093        respectively) to potentiate trehalose uptake.    -   The gene encoding cellobiose-specific PTS system IIC component        (Gene ID: 4796893), ptcC, was disrupted (tga at codon position        30 of 446; tga30). This mutation ascertains trehalose retention        after accumulation.    -   A gene encoding a fusion of usp45 secretion leader (SSusp45)        with the pins gene, encoding human proinsulin (PINS; UniProt:        P01308, amino acids 25-110) is positioned downstream of the        glyceraldehyde 3-phosphate dehydrogenase gene (gapB; Gene ID:        4797877), to allow expression and secretion of PINS. The pins        expression unit was transcriptionally and translationally        coupled to gapB by use of IRrpmD.

All genetic traits of sAGX0407 reside on the bacterial chromosome. Thegenetic background of sAGX0407 provides:

-   -   Constitutive secretion of PINS and hIL-10.    -   Strict dependence on exogenously added thymidine for growth and        survival.    -   The capacity to accumulate and retain trehalose and so acquire        the capacity to resist bile acid toxicity.

FIG. 18 shows a schematic overview of relevant genetic loci of sAGX0407as described: trePTS, ΔtrePP; otsB; ptcC-; gapB>>pins and ΔthyA, hIL-10with indication of the relevant oligonucleotide binding sites (oAGXno),EcoRI restriction site, (/truncated/) genetic characters, intergenicregions (IR), PCR amplification product sizes (bp).

trePTS, ΔtrePP

Deletion of trehalose-6-phosphate phosphorylase gene (trePP; Gene ID:4797140); Insertion of the constitutive promoter of the HU-likeDNA-binding protein gene (PhllA; Gene ID: 4797353) to precede theputative phosphotransferase genes in the trehalose operon (trePTS;llmg_0453 and llmg_0454; ptsI and ptsII; Gene ID: 4797778 and Gene ID:4797093 respectively), insertion of the intergenic region preceding thehighly expressed L. lactis MG1363 50S ribosomal protein L30 gene (rpmD;Gene ID: 4797873) in-between ptsI and ptsII (FIGS. 13A, 13B, and 13C).

otsB

Insertion of trehalose-6-phosphate phosphatase gene (otsB; Gene ID:1036914) downstream of unidentified secreted 45-kDa protein gene (usp45;Gene ID: 4797218). Insertion of the intergenic region preceding thehighly expressed L. lactis MG1363 50S ribosomal protein L30 gene (rpmD;Gene ID: 4797873) between usp45 and otsB. (FIGS. 14A and 14B).

ptcC—

Insertion of tga at codon position 30 of 446 (tga30), alongside with theintroduction of an EcoRI restriction site, to disrupt the gene encodingcellobiose-specific PTS system IIC component (ptcC; Gene ID: 4796893).(FIGS. 15A and 15B)

gapB>Pins

Insertion of a gene encoding a fusion of usp45 secretion leader(SSusp45) with the pins gene, encoding human proinsulin (PINS; UniProt:P01308, amino acids 25-110), downstream of the highly expressedglyceraldehyde 3-phosphate dehydrogenase gene (gapB; Gene ID: 4797877).Insertion of the intergenic region preceding the highly expressed L.lactis MG1363 50S ribosomal protein L30 gene (rpmD; Gene ID: 4797873)between gapB and pins (FIGS. 12A, 12B, and 16)

ΔthyA, hIL-10

Deletion of thymidylate synthase gene (thyA; Gene ID: 4798358).Downstream of the constitutive promoter of the HU-like DNA-bindingprotein gene (PhllA; Gene ID: 4797353), insertion of a gene encoding afusion of SSusp45 with the hil-10 gene, encoding human interleukin-10(hIL-10; UniProt: P22301, aa 19-178, variant P2A, Steidler et al., Nat.Biotechnol. 2003, 21(7): 785-789) is inserted to allow expression andsecretion of hIL-10 (FIG. 17).

Example 2 Treatment of Diabetes in Mice

NOD mice were screened for the onset of diabetes by evaluating glucoselevels in urine (Diastix® Reagent strips, Bayer, Leverkusen, Germany)and venous blood (AccuCheck®, Roche Diagnostics, Vilvoorde, Belgium).Mice were diagnosed as diabetic when having glucosuria and twoconsecutive blood glucose measurements exceeding 200 mg/dl. Upondiabetes determination, NOD or NOD transgenic mice were treated for 5consecutive days intravenously (i.v.) (day 0-4; 2.5 μg/mouse) withhamster anti-mouse CD3 antibodies (clone 145-2C11, BioXCell, WestLebanon, N.H.). This therapy was given in combination with oraladministration of either plasmid-driven or clinical-grade L. lactisstrains (2×10⁹ cfu) 5 times per week during 6 weeks. Control mice wereleft untreated. Individual blood glucose concentrations at the start oftreatment were recorded. Mice were tested 3 times weekly for theirweight and blood glucose status. Remission was defined as the absence ofglucosuria and a return to normal blood glucose concentrations.Experimental animals were sacrificed immediately or long after stoppingtherapy (6 or 14 weeks after treatment initiation). Peripheral blood,lymph organs and pancreas were harvested, and single cells were assessedfor phenotyping as described in the Supplemental Research Design andMethods. Mice were removed from the study prior to the 14-week endpointwhen blood glucose concentrations exceeded 600 mg/dl in two consecutivemeasurements.

Glucose Tolerance Test

One or two weeks prior to sacrifice intraperitoneal glucose tolerancetests (IPGTT) were performed. Mice were fasted for 16 hours, injectedintraperitoneally (i.p.) with glucose (2 g/kg) and blood glucoseconcentrations were measured at 0, 15, 30, 60, 90 and 120 minutes.

IAA Measurement

Heparinized plasma was collected from new-onset diabetic NOD mice beforetreatment randomization and at therapy discontinuation, and IAAs wereanalyzed at the UF Department of Pathology, Immunology and LaboratoryMedicine, College of Medicine, Gainesville, Fla., as described in RobertS. et al., Diabetes 2014, 63: 2876-2887.

In Vivo Blocking of CTLA4 and TGF-β

Mice tolerized by L. lactis-based therapy were injectedintraperitoneally (i.p.) after therapy withdrawal with blockingantibodies against CTLA4 (clone UC10-4F10, Bioceros) and TGF-β (clone1D11.16.8, BioXCell) in the following dose regimen: 250 μg at day 1 and3 and then 100 μg at day 6, 8, 10, 13 and 18 for CTLA4; 200 μg 3 timesper week during 3 weeks for TGF-β. Blood glucose concentrations weremeasured daily up to 25 days after first injection.

Adoptive Transfer of Diabetes.

To assess the diabetogenic potential of Teff cells, total T cells fromspleen (1×10⁷ cells) of new-onset diabetic controls, responders andnon-responders of L. lactis-based therapy were transferred i.v.(intravenously) into the tail veins of 6- to 8-week-old immune-deficientNOD-scid mice. Recipient mice were monitored twice weekly for thedevelopment of diabetes up to 100 days post-cell transfer.

DT-Mediated Depletion of Foxp3⁺ T Cells in NOD.Foxp3.DTR Mice

NOD.Foxp3.DTR mice (expressing the human diphtheria toxin receptor (DTR)under the control of Foxp3 transcriptional control elements) allow forthe depletion of Foxp3⁺ T cells upon DT (diphtheria toxin)administration. See, e.g., Feuerer M. et al., How punctual ablation ofregulatory T cells unleashes an autoimmune lesion within the pancreaticislets. Immunity 2009; 31:654-664. For Treg depletion, NOD.Foxp3.DTRmice (unmanipulated or tolerized after stopping the L. lactis-basedtherapy) were injected i.p. with 40 μg/kg bodyweight of DT (Sigma) ondays 1, 2, 4, and 7 and examined on day 8. Following DT injections,weight, urine and blood glucose status of mice were monitored. Foxp3⁺ Tcells were monitored in peripheral blood and pancreas by flow cytometryand histology respectively as described. See, e.g., Tian L. et al.,Foxp3(+) regulatory T cells exert asymmetric control over murine helperresponses by inducing Th2 cell apoptosis. Blood 2011; 118:1845-1853.

FOXP3-Inhibitory Peptide P60 in Combination with L. lactis-Based Therapy

P60 (a 15-mer synthetic peptide that can bind to and block FOXP3, i.p.50 μg/dose daily, up to 14 doses) was given either at start of the L.lactis-based therapy, as previously described. See, e.g., Casares N. etal., A peptide inhibitor of FOXP3 impairs regulatory T cell activity andimproves vaccine efficacy in mice. Journal of Immunology 2010,185:5150-5159.

Histology of Pancreas and Insulitis Grading

Six-μm sections from formalin-fixed paraffin-embedded pancreas tissueswere cut and collected 100-μm apart, then stained with hematoxylineosin. Islets were observed under light microscopy at 20× or 40×,enumerated and graded by an independent investigator in blinded fashion.At least 25 islets per pancreatic sample were scored for isletinfiltration as follows: 0, no infiltration; 1, peri-insulitis; 2,islets with lymphocyte infiltration in less than 50% of the area, 3,islets with lymphocyte infiltration in more than 50% of the area orcompletely destroyed.

Islet-Resident Foxp3⁺ T Cell Detection

Pancreas tissues were snap-frozen in 2-methyl-butane 99% and cut into12-μm tissue sections. Foxp3⁺ T cell detection was performed asdescribed. See, e.g., Takiishi T. et al., Reversal of autoimmunediabetes by restoration of antigen-specific tolerance using geneticallymodified Lactococcus lactis in mice. J. Clin. Invest. 2012,122:1717-1725.

Statistics

All data were analyzed using GraphPad Prism 6 (Graphpad Prism, La Jolla,Calif.). Survival curves were computed with Kaplan-Meier test andcompared with log-rank test. Groups were analyzed by ANOVA(non-parametric Kruskal-Wallis test) with Dunn's multiple comparison orwith Mann-Whitney U test, as appropriate. Error bars represent SEM.Unless otherwise indicated, differences are not significant (ns).*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Bacteria and Media

L. lactis-pT1NX is an MG1363 strain containing the empty vector pT1NX,and served as control. The plasmid-driven L. lactis strain (sAGX0328secreting plasmid-encoded human PINS and chromosomally-integrated IL10)was cultured as previously described. See, e.g., Takiishi T. et al., J.Clin. Invest. 2012, 122:1717-17253. As growth and survival ofthyA-deficient L. lactis strains depends on the presence of thymidine inthe growth medium, the clinical-grade L. lactis (secretingchromosomally-integrated human PINS and IL10) was cultured in GM17T,i.e. M17 broth (BD, Franklin Lakes, N.J.), supplemented with 0.5%glucose (Merck KGaA, Darmstadt, Germany) and 200 gtM thymidine (Sigma,St. Louis, Mo.). For intragastric inoculations, stock suspensions werediluted 1000-fold in growth media and incubated for 16 hours at 30° C.,reaching a saturation density of 2×10⁹ cfu/ml. Bacteria were harvestedby centrifugation and concentrated 10-fold in BM9 medium. Treatmentdoses consisted of 100 μl of this bacterial suspension. For intragastricinoculations, stock suspensions were diluted 1000-fold in growth mediaand incubated for 16 hours at 30° C., reaching a saturation density of2×10⁹ cfu/ml. Bacteria were harvested by centrifugation and concentrated10-fold in BM9 medium. Treatment doses consisted of 100 μl of thisbacterial suspension.

Flow Cytometry

Peripheral blood and specified organs were harvested 6 weeks aftertreatment initiation, processed and incubated with fluorochrome-labeledantibodies or matching isotype controls for flow cytometric analysis.Tregs were stained with anti-mouse CD3 (145-2C11), CD4 (GK1.5), CD8a(53-6.7), CD25 (PC61.5 or 7D4 (BD, Erembodegem, Belgium)), and FR4(eBio12A5) (all from eBioscience, San Diego, Calif., unless specified)for 20 min on ice. Intracellular staining antibodies against Foxp3(FJK-16s) and CTLA4 (UC10-4B9) were from eBioscience and used accordingto the manufacturer's instructions. Naïve, effector memory and centralmemory T cells were determined by staining with anti-mouse CD3(145-2C11), CD4 (GK1.5), CD8a (53-6.7), CD44 (IM7), CD62L (MEL-14), CD69(H1.2F3), and CCR7 (4B12). Cells were analyzed in a Gallios™ flowcytometer with Kaluza (Beckman Coulter, Suarlée, Belgium) or FlowJosoftware (Treestar, Ashland, Oreg.).

In Vitro Suppressor Assays

Pathogenic CD4⁺CD25⁻ Teff cells were isolated from spleen cells of10-week old NOD mice by negative selection using antibodies to CD25,CD8α, B220, CD11c, CD11b, MHC class II, and sheep anti-rat IgG Dynabeads(Invitrogen, Merelbeke, Belgium). CD4⁺CD25⁺Foxp3 and CD4⁺CD25⁻Foxp3⁺Tregs were isolated from pooled lymph node and spleen cells ofNOD.Foxp3.hCD2 mice (harboring a human CD2-CD52 fusion protein, alongwith an intra-ribosomal entry site, into the 3′ untranslated region ofthe endogenous foxp3 locus). See, e.g., Culina S. et al., Clin. Dev.Immunol. 2011; 2011:286248. Briefly, cell samples were passed through a70-μm cell strainer and suspended in RPMI1640 medium, centrifuged for 5min at 1,500 rpm and suspended in RPMI1640 medium. The resultant cellsuspension was counted, washed and first depleted of CD8α⁺, B220⁺,CD11c⁺, CD11b⁺ and MHC class⁺, and adherent cells by panning and stainedwith biotin-conjugated anti-CD4 and anti-hCD2 antibodies. Cells werethen incubated with anti-biotin microbeads (Miltenyi Biotec B. V.,Leiden, The Netherlands) and separated on LS or MS columns (MiltenyiBiotech). The resulting hCD2⁺ or hCD2⁻ cells were further purified withanti-CD25 antibodies. In vitro polyclonal suppressor assays wereconducted. Cytokines (IFN-γ, IL10 and TGF-β) were measured in cell-freesupernatants by multiplex immunoassay (Mesoscale Discovery, Rockville,Mass.) or flow cytometric bead array (Bender MedSystems FlowCytomix™,eBioscience) as described. See, e.g., Ludvigsson J. et al., GAD65antigen therapy in recently diagnosed type 1 diabetes mellitus. N. Engl.J. Med. 2012, 366:433-442.

Blocking antibodies against CTLA4 (UC10-4F10), IL10 (clone JES5-2A5,BioXCell), and TGF-β (clone 1D11.16.8, which neutralizes all threemammalian TGF-β isoforms (β1, β2, and β3), BioXCell) were added tocultures at a concentration of 10 μg/ml.

Results

A Clinical-Grade Self-Contained L. lactis Vaccine Combined with Low-DoseAnti-CD3 Stably Reversed New-Onset Diabetes, Preserved ResidualBeta-Cell Function and Halted Insulitis Progression in NOD Mice.

Using a clinical-grade self-containing L. lactis strain secreting humanPINS along with IL10 in combination with sub-therapeutic doses ofanti-CD3 antibodies, 66% (23 out of 35) of mice reverted tonormoglycemia for at least 14 weeks after disease onset, which wasstatistically significantly superior to 43% of mice treated by anti-CD3alone (FIG. 1A). Animals left untreated (n=20) or treated with the emptyvector bacterial strain L. lactis-pT1NX (n=9) remained hyperglycemic andwere sacrificed when 20% of their starting body weight was lost.Monotherapy with the clinical-grade or plasmid-driven L. lactis strainsecreting PINS and IL-10 was significantly less effective than thecombination with anti-CD3 (0% (n=8) and 17% (n=8) respectively) (FIG.1A).

During follow up, new-onset diabetic controls, mice protected by L.lactis-based therapy, and mice not protected by such therapy, weresubjected to IPGTT and sacrificed 6 weeks after treatment initiation atwhich time their pancreas tissues were assessed by histology. Only inthe successfully treated animals, residual beta-cell function (i.e.assessed as area under glucose tolerance curve (AUCglucose)) waspreserved and smaller proportions of islets had severe insulitis (FIG.1B). Of interest, at the end of the combination therapy no difference inthe severity of insulitis was observed between responders andnon-responders (FIG. 1C).

Starting Glycemia and IAA Positivity Predict Therapeutic Success of L.lactis-Based Therapy.

No influence of age or gender of mice was observed on therapeuticsuccess of the L. lactis-based therapy. However, glycemic concentrationsat the beginning of therapy predicted success with 82% of mice startingwith a glycemia below 350 mg/dl cured (n=22), in comparison to 38% ofmice with a starting glycemia above 350 mg/dl (n=13) (FIG. 2A). Inaddition, positivity for IAAs at entry seemed to correlate withtherapeutic success (FIG. 2B). Interestingly, mice with blood glucoseconcentrations<350 mg/dl and IAA positivity at therapy start had aclearly superior diabetes remission rate (89%, n=8) than mice with bloodglucose levels>350 mg/dl and being IAA negative (33%; n=5; P=0.07).(FIG. 2C). Moreover, the L. lactis-based therapy significantly decreasedIAA levels, particularly in mice responsive to the therapy (FIG. 2D).

L. lactis-Based Therapy Induces Higher Levels of Foxp3⁺ T Cells withRegulatory Capacity but No Changes in Teff Cells.

The mechanisms underlying disease remission induced by the L.lactis-based treatment were investigated by dissociating between thetherapeutic immune effects in mice responsive or not to theintervention. We found that the percentages of CD4⁺Foxp3⁺ (both CD25⁺and CD25⁻) T cells observed in the peripheral blood (FIG. 3A), thepancreatic draining lymph nodes (FIG. 3B), and the pancreas (FIG. 3C)were significantly higher in mice treated with the L. lactis-basedtherapy in comparison to untreated controls. Interestingly, in thepancreatic draining lymph nodes and pancreas, but not in peripheralblood, the increased frequency of CD4⁺Foxp3⁺ T cells was less pronouncedin responders than non-responders. Using multicolor flow cytometry, weidentified that most CD4⁺Foxp3⁺ Tregs were positive for CTLA4 and thatthe expression of this inhibitory marker was significantly higher inpancreatic draining lymph nodes (for both responders and non-responders)and pancreas (only for responders) of treated mice compared to untreatedcontrols (FIGS. 8B and 8C). Of interest, no differences in thepercentages of CD4⁺Foxp3⁺CTLA4⁺ T cells were observed in the peripheralblood of treated mice compared to untreated controls (FIG. 3D). Thepercentages of naïve (CD44⁻CD62L⁺CCR7⁺), effector memory(CD44⁺CD62L⁻CCR7⁻) and central memory (CD44⁺CD62L⁺CCR7⁺) CD4⁺ T cellswere not altered in any recipient group with respect to therapeuticsuccess or failure. Transfer of splenocytes from responders andnon-responders of L. lactis-based treatment caused diabetes in NOD-scidrecipients with similar disease kinetics as transfer of splenocytesisolated from untreated new-onset diabetic controls, suggesting thatcirculating diabetogenic cells were not depleted from treated mice (FIG.9).

Diabetes Reversal Induced by L. lactis-Based Therapy is Accompanied byand Depends on the Generation of Functional Foxp3⁺ Tregs.

Using NOD.Foxp3.hCD2 mice treated by L. lactis-based therapy, we couldisolate CD4⁺CD25⁺Foxp3⁺ T cells for functional in vitro studies, inwhich they suppressed proliferation, CD69 activation and IFN-γproduction of pathogenic CD4⁺CD25⁻ Teff cells. These Tregs producedIL-10 (and TGF-β) when they were co-cultured and stimulated withanti-CD3 antibody in the presence of splenic antigen-presenting cells(APCs) isolated from NOD-scid γc−/− mice (FIG. 4). No difference inregulatory capacity of CD4⁺CD25⁻Foxp3⁺ T cells was seen between therapyresponders and non-responders.

Addition of anti-CTLA4 Ig (clone UC10-4F10) or a TGF-β neutralizingantibody (clone 1D11.16.8) significantly reduced the suppression by theCD4⁺CD25⁺Foxp3⁺ T cells (FIG. 5A), suggesting that CD4⁺CD25⁺Foxp3⁺ Tregsof cured mice inhibit Teff proliferation via a CTLA4- andTGF-β-dependent fashion in vitro. Adding anti-IL10 (clone JES5-2A5) didnot alter the direct suppressive effect of the Tregs. On the other hand,these regulatory mechanisms were not demonstrated with theCD4⁺CD25⁻Foxp3⁺ T cell fraction from therapy responders andnon-responders (FIG. 5B). Treating stably cured mice in vivo with acombination of anti-CTLA4 Ig (clone UC10-4F10) and anti-TGF-β (clone1D11.16.8) led to diabetes recurrence in 2 out of 5 mice (FIG. 5C).

Finally, we investigated whether the therapeutic success of L.lactis-based therapy was depended on the presence and functionality ofFoxp3⁺ T cells. For this, new-onset diabetic NOD mice weresimultaneously treated with the L. lactis-based therapy and theFOXP3-inhibitory peptide P60 for a period of 14 days (FIG. 6A).Interestingly, none of the mice (n=6) developed normoglycemia, whilemice treated with the L. lactis-based therapy and vehicle (n=1) had a60% diabetes remission rate, indicating that Tregs are crucial forinduction of therapy-induced tolerance (FIG. 6B).

New-onset (spontaneously) diabetic NOD.Foxp3.DTR mice were treated withthe L. lactis-based therapy and after stable diabetes reversal wasobserved, Foxp3⁺ T cells were eliminated using DT as described in thescheme depicted in FIG. 7A. First, we established in unmanipulatedNOD.Foxp3.DTR mice that the selected DT regimen eliminated over 90% ofCD4⁺Foxp3⁺ T cells, with the remaining Tregs expressing low or no CD25,in the peripheral blood within 3 days after first DT injection (FIGS.10A-C). A progressive repopulation of these cells started from day 5after first DT injection as has been reported for several Foxp3.DTRstrains. See, e.g., Kim J. M. et al., Nat. Immunol. 2007, 8:191-197;Suffner J. et al., J. Immunol. 2010, 184:1810-1820; and Mayer C. T. etal., Immun. Inflamm. Dis. 2014, 2:162-165. This DT regimen alsodramatically decreased the amount of Foxp3⁺ T cells residing in thepancreas, consequently leading to the development of autoimmune diabetes(FIGS. 10B and 10D). Next, comparable to wild-type NOD mice, the L.lactis-based treatment induced autoimmune diabetes remission in 57% ofNOD.Foxp3.DTR mice (4 out of 7 mice) (FIGS. 7A and 7B). Transient Foxp3⁺T cell depletion resulted in a complete reversal to the diabetic statein all mice (n=4) that were initially cured by the therapy, as evidencedby the reappearance of glucosuria along with severe hyperglycemiastarting from day 2 after first DT injection (FIG. 7B). This breach ofimmune tolerance to insulin-producing beta-cells was also accompanied bythe induction of severe insulitis (FIG. 7C) and the ablation of theislet-resident Foxp3⁺ Treg pool (FIG. 7D). Collectively, these datademonstrated that the therapeutic effect from the L. lactis-basedintervention depended on the presence and functionality of Foxp3⁺ Tregs.

DISCUSSION

Oral tolerance as a means of intervention to arrest disease has beenexplored in animal models of autoimmune disease including T1D. See,e.g., Commins S P, Pediatr. Clin. North. Am. 2015; 62:1523-1529.

In this example, an oral clinical-grade self-containing L. lactis strainsecreting chromosomally-integrated human PINS and human IL-10 wasprepared. When combined with a short course of sub-therapeutic doses ofanti-CD3, the intervention was safe and effective in inducing long-termnormoglycemia in new-onset diabetic mice. Initial blood glucoseconcentrations (<350 mg/dl) in addition to IAA positivity at diseaseonset were predictors of therapeutic outcome, while preservation ofresidual beta-cell function and decline in IAA positivity were markersof therapeutic success.

Thus, some degree of residual beta-cell mass will be necessary fortherapeutic success when intervening at the moment of diabetesdiagnosis, namely when dysglycemia is present.

By dissociating between the immune effects of the L. lactis-basedintervention in mice responsive or not to the therapy, the nature androle of the immune processes accompanying the treatment was furthercharacterized. The L. lactis-based therapy induced suppressiveIL-10-secreting CD4⁺Foxp3⁺ (both CD25⁺ and CD25⁻) T cells in thepancreatic draining lymph nodes and pancreas of responders and even moreso of non-responders, suggesting enhanced recruitment of Tregs to theinflamed target tissues. In the periphery, the frequency of CD4⁺Foxp3⁺ Tcells was also increased in treated mice compared to untreated controls,pointing towards a possible use this cell population as immune marker.Interestingly, the frequency of CTLA4⁺ T-cells among various Tregsubsets was significantly higher in the pancreas of combinationtherapy-treated responder mice compared to new-onset diabetic mice andin contrast to combination therapy-treated non-responder mice. Nodifference was seen in the degree of insulitis between responder andnon-responder mice, suggesting alterations in lymphocyte subsets otherthan Tregs.

Peripheral Tregs can use different regulatory mechanisms according totheir environmental milieu and stimulatory conditions. See, e.g., ListonA. et al., Nature Reviews Immunology 2014; 14:154-165. In theseexperiments, CTLA4 and TGF-β were important for the regulatory activityof therapy-expanded CD4⁺CD25⁺Foxp3⁺ T cells in vitro and partially invivo, while IL-10 was not.

Although IL-10 seemed to be a good marker for the identification of ourL. lactis-based therapy-induced Tregs, others have demonstrated thatanti-IL-10 antibodies did not abrogate established tolerance in vivo.See, e.g., Fowler S. and Powrie F, European Journal of Immunology 2002,32:2997-3006.

As in mice, human Tregs are defined by having a suppressive phenotypeendowed by high and sustained expression of the transcription factorFoxp3 (see, e.g., Hori S. et al., Science 2003; 299:1057-1061) and lossof function/mutation in the Foxp3 gene leads to severe fatal autoimmunedisorders (see, e.g., Kim J. M., et al., Nature Immunology 2007;8:191-197; and Mayer C. T. et al., European Journal of Immunology 2014,44:2990-3002).

Specific inhibition of Treg functionality by the P60 peptide (see, e.g.,Casares N. et al., Journal of Immunology 2010; 185:5150-5159) at thestart of L. lactis-based therapy impaired the induction oftherapy-induced tolerance. Moreover, transient depletion of Foxp3⁺ Tregsfrom therapy-tolerized NOD.Foxp3. DTR mice was sufficient to inducecomplete disease relapse in all animals, demonstrating that the presenceof Foxp3⁺ T cells was important to maintain therapeutic tolerance andcontrol pathogenic Teff cells which were still present in miceresponsive to L. lactis-based therapy.

The current data demonstrate that combining a clinical-gradeself-containing L. lactis secreting human PINS and human IL-10 withlow-dose anti-CD3 increased the frequency of diabetes reversal comparedto anti-CD3 mono-therapy. Both therapy responders and non-responders hadincreased frequencies of CD4⁺Foxp3⁺ T cells, suggesting that immuneeffects induced by the L. lactis-based therapy occurred in eachindividual recipient, but that therapeutic success (defined as return tostable normoglycemia) depended on other parameters, such as functionalbeta-cell mass still present at disease onset. Therapeutic success wasfurther correlated with starting glycemia. Next to initial blood glucoseconcentrations at entry, also IAA levels predicted outcome of this L.lactis-based therapy using PINS as antigen. The current datademonstrates that Foxp3⁺ Tregs were essential to induce and maintainactive tolerance and control diabetogenic immune responses in tolerizedmice. These findings provide tools for testing interventions in humans:a clinical-grade self-containing L. lactis secreting islet antigen(s),biomarkers for predicting therapeutic success, and the demonstrationthat the induction of Foxp3⁺ T cells is the basis of the L. lactis-basedtherapy.

While some embodiments have been shown and described herein, suchembodiments are provided by way of example only. Numerous variations,changes, and substitutions will occur to those skilled in the artwithout departing from the invention. It should be understood thatvarious alternatives to the embodiments of the invention describedherein will be employed in practicing the invention.

Exemplary Embodiments

1. A lactic acid bacterium (LAB) comprising an exogenous nucleic acidencoding interleukin-10 (IL-10), and an exogenous nucleic acid encodingproinsulin (PINS), wherein said exogenous nucleic acid encoding IL-10and said exogenous nucleic acid encoding PINS are chromosomallyintegrated.

-   2. The LAB of embodiment 1, wherein said LAB is a Lactococcus    species bacterium.-   3. The LAB of embodiment 2, wherein said LAB is Lactococcus lactis.-   4. The LAB of embodiment 3, wherein said LAB is Lactococcus lactis    subspecies cremoris.-   5. The LAB of embodiment 4, wherein said LAB is Lactococcus lactis    strain MG1363.-   6. The LAB of any one of embodiments 1 to 5, wherein said IL-10 is    human IL-10 (hIL-10).-   7. The LAB of embodiment 6, wherein said hIL-10 when secreted as a    mature hIL-10 without a signal peptide comprises alanine (Ala)    instead of proline (Pro) at position 2.-   8. The LAB of any one of embodiments 1 to 7, wherein said IL-10 has    an amino acid sequence at least 95% identical to SEQ ID NO: 1.-   9. The LAB of any one of embodiments 1 to 8, wherein said exogenous    nucleic acid encoding IL-10 has a nucleotide sequence at least 95%    identical to SEQ ID NO: 2.-   10. The LAB of any one of the preceding embodiments, wherein said    PINS is human PINS (hPINS).-   11. The LAB of any one of the preceding embodiments, wherein said    PINS has an amino acid sequence at least 95% identical to SEQ ID NO:    3.-   12. The LAB of any one of the preceding embodiments, wherein said    exogenous nucleic acid encoding PINS has a nucleotide sequence at    least 95% identical to SEQ ID NO: 4.-   13. The LAB of any one of the preceding embodiments, wherein said    LAB constitutively expresses said IL-10.-   14. The LAB of embodiment 13, wherein said LAB secretes said IL-10.-   15. The LAB of any one of the preceding embodiments, wherein said    LAB constitutively expresses said PINS.-   16. The LAB of embodiment 15, wherein said LAB secretes said PINS.-   17. The LAB of any one of the preceding embodiments, wherein said    LAB constitutively expresses and secretes said IL-10 and said PINS.-   18. The LAB of any one of the preceding embodiments, wherein said    exogenous nucleic acid encoding IL-10 is positioned 3′ of an hllA    promoter (PhllA).-   19. The LAB of embodiment 18, wherein said PhilA is a Lactococcus    lactis PhllA.-   20. The LAB of embodiment 18, wherein said exogenous nucleic acid    encoding IL-10 is transcriptionally regulated by said PhllA.-   21. The LAB of any one of the preceding embodiments, wherein said    LAB comprises an expression cassette comprising an hllA promoter, an    IL-10 secretion sequence, and said exogenous nucleic acid encoding    IL-10.-   22. The LAB of embodiment 21, wherein said expression cassette is    chromosomally integrated.-   23. The LAB of embodiment 22, wherein said expression cassette is    chromosomally integrated at a thyA locus.-   24. The LAB of any one of embodiments 21 to 23, wherein said IL-10    secretion sequence is a nucleotide sequence encoding a secretion    leader of unidentified secreted 45-kDa protein (Usp45) (SSusp45).-   25. The LAB of any one of the preceding embodiments, wherein said    exogenous nucleic acid encoding PINS is positioned 3′ of a gapB    promoter.-   26. The LAB of embodiment 25, wherein said exogenous nucleic acid    encoding PINS is transcriptionally regulated by said gapB promoter.-   27. The LAB of embodiment 25 or 26, wherein said LAB comprises a    first polycistronic expression cassette comprising a gapB promoter    positioned 5′ of a gapB gene, a PINS secretion sequence, and said    exogenous nucleic acid encoding PINS.-   28. The LAB of embodiment 27, wherein said LAB further comprises an    intergenic region between said gapB gene and said PINS secretion    sequence.-   29. The LAB of embodiment 28, wherein said intergenic region is rpmD    having SEQ ID NO: 8 or SEQ ID NO: 9.-   30. The LAB of any one of embodiments 27 to 29, wherein said gapB    promoter and said gapB gene are endogenous to said LAB.-   31. The LAB of any one of embodiments 27 to 30, wherein said first    polycistronic expression cassette is chromosomally integrated.-   32. The LAB of any one of embodiments 27 to 31, wherein said PINS    secretion sequence is a nucleotide sequence encoding SSusp45.-   33. The LAB of embodiment 32, wherein said SSusp45 has a nucleotide    sequence of SEQ ID NO: 11 or SEQ ID NO: 12.-   34. The LAB of any one of the preceding embodiments, wherein said    LAB further comprises an exogenous nucleic acid encoding a    trehalose-6-phosphate phosphatase.-   35. The LAB of embodiment 34, wherein said trehalose-6-phosphate    phosphatase is Escherichia coli OtsB.-   36. The LAB of embodiment 34 or 35, wherein said exogenous nucleic    acid encoding a trehalose-6-phosphate phosphatase is chromosomally    integrated.-   37. The LAB of embodiment 36, wherein said exogenous nucleic acid    encoding a trehalose-6-phosphate phosphatase is chromosomally    integrated 3′ of unidentified secreted 45-kDa protein gene (usp45).-   38. The LAB of embodiment 37, wherein said LAB comprises a second    polycistronic expression cassette comprising a usp45 promoter, said    usp45, and said exogenous nucleic acid encoding a    trehalose-6-phosphate phosphatase.-   39. The LAB of embodiment 38, wherein said second polycistronic    expression cassette further comprises an intergenic region between    said usp45 and said exogenous nucleic acid encoding a    trehalose-6-phosphate phosphatase.-   40. The LAB of embodiment 39, wherein said intergenic region is rpmD    having SEQ ID NO: 8 or SEQ ID NO: 9.-   41. The LAB of any one of the preceding embodiments, wherein a    trehalose-6-phosphate phosphorylase gene (trePP) is disrupted or    inactivated in said LAB.-   42. The LAB of embodiment 41, wherein said trePP is inactivated by    removing said trePP or a fragment thereof.-   43. The LAB of embodiment 41, wherein said trePP is disrupted by    insertion of a stop codon.-   44. The LAB of any one of embodiments 41-43, wherein said LAB lacks    trePP activity.-   45. The LAB of any one of the preceding embodiments, wherein a    cellobiose-specific PTS system IIC component gene (ptcC) is    disrupted or inactivated in said LAB.-   46. The LAB of embodiment 45, wherein said ptcC is disrupted by    inserting a stop codon.-   47. The LAB of embodiment 45, wherein said ptcC is inactivated by    removing said ptcC or a fragment thereof.-   48. The LAB of any one of embodiments 45 to 47, wherein said LAB    lacks ptcC activity.-   49. The LAB of any of the preceding embodiments, wherein said LAB    further comprises one or more genes encoding one or more trehalose    transporter(s).-   50. The LAB of embodiment 49, wherein said one or more genes    encoding one or more trehalose transporter(s) is endogenous to said    LAB.-   51. The LAB of embodiment 49 or 50, wherein said LAB overexpresses    said one or more trehalose transporter(s).-   52. The LAB according to any one of embodiments 49 to 51, wherein    said one or more genes encoding one or more trehalose transporter(s)    is/are positioned 3′ of an hllA promoter (PhilA).-   53. The LAB of embodiment 52, wherein said one or more genes    encoding one or more trehalose transporter(s) is/are    transcriptionally regulated by said PhllA.-   54. The LAB of any one of embodiments 47 to 51, wherein said one or    more genes encoding one or more trehalose transporter(s) is/are    selected from the group consisting of llmg_0453, llmg_0454, and any    combination thereof.-   55. The LAB of embodiment 54, wherein said one or more genes    encoding one or more trehalose transporter(s) comprises two genes    encoding two trehalose transporters, wherein an intergenic region is    located between said two genes.-   56. The LAB of embodiment 55, wherein said intergenic region is rpmD    having SEQ ID NO: 8 or SEQ ID NO: 9.-   57. A composition comprising the LAB of any one of the preceding    embodiments.-   58. A pharmaceutical composition comprising the LAB of any one of    embodiments 1 to 56, and a pharmaceutically acceptable carrier.-   59. The LAB of any one of embodiments 1 to 56, the composition of    embodiment 57, or the pharmaceutical composition of embodiment 58,    for use in the treatment of type-1 diabetes (T1D).-   60. The LAB of any one of embodiments 1 to 56, the composition of    embodiment 57, or the pharmaceutical composition of embodiment 58,    for use in the preparation of a medicament for the treatment of T1D.-   61. The LAB, the composition, or the pharmaceutical composition of    embodiment 60, wherein said T1D is recent-onset T1D.-   62. A method for the treatment of T1D in a subject in need thereof    comprising administering to said subject a therapeutically effective    amount of the LAB of any one of embodiments 1 to 56, the composition    of embodiment 57, or the pharmaceutical composition of embodiment    58.-   63. The method of embodiment 62, wherein said T1D is recent-onset    T1D.-   64. The method of embodiment 62 or 63, wherein said subject is a    human.-   65. The method of any one of embodiments 62 to 64, wherein said    method further comprises administering a therapeutically effective    amount of an anti-CD3 antibody to said subject.-   66. The method of embodiment 65, wherein said anti-CD3 antibody is a    monoclonal antibody.-   67. The method of embodiment 65 or 66, wherein said anti-CD3    antibody is otelixizumab or teplizumab.-   68. The method of any one of embodiments 65 to 67, wherein said    antibody is administered to said subject using a co-therapeutic    regimen.-   69. The method of any one of embodiments 65 to 68, wherein said    administering said LAB and said administering said anti-CD3    antibody:    -   (i) reduces an amount of insulin the subject needs to maintain a        normal blood glucose level, when compared to a corresponding        subject treated with anti-CD3 antibody alone, or compared to a        corresponding subject not being administered said LAB and said        anti-CD3 antibody;    -   (ii) preserves initial beta-cell function in said subject for at        least about 2 months as measured by C-peptide level;    -   (iii) maintains normal glycemia in the subject for at least        about 2 months;    -   (iv) maintains a normal hemoglobinA1c (HbA1c) level in said        subject for at least about 2 months; or    -   (v) any combination thereof.-   70. The method of any one of embodiments 62 to 69, wherein the    method further comprises measuring insulin autoantibody (IAA) in    said subject.-   71. A method for preparing a genetically modified LAB comprising (i)    contacting an LAB with an exogenous nucleic acid encoding IL-10;    and (ii) contacting said LAB with an exogenous nucleic acid encoding    PINS, wherein said exogenous nucleic acid encoding IL-10, and said    exogenous nucleic acid encoding PINS are chromosomally integrated.-   72. The method of embodiment 71, wherein said exogenous nucleic acid    encoding IL-10 and said exogenous nucleic acid encoding PINS are    chromosomally integrated using homologous recombination.-   73. The method of embodiment 71 or 72, wherein said contacting said    LAB with an exogenous nucleic acid encoding IL-10 occurs prior to    said contacting said LAB with an exogenous nucleic acid encoding    PINS.-   74. The method of embodiment 71 or 72, wherein said contacting said    LAB with an exogenous nucleic acid encoding IL-10 occurs subsequent    to said contacting said LAB with an exogenous nucleic acid encoding    PINS.-   75. The method of any one of embodiments 71 to 74, further    comprising combining a culture of said genetically modified LAB with    at least one stabilizing agent to form a bacterial mixture.-   76. The method of embodiment 75, further comprising removing water    from said bacterial mixture forming a dried composition.-   77. The method of embodiment 76, comprising freeze-drying said    bacterial mixture to form a freeze-dried composition.-   78. The method of embodiment 77, wherein said at least one    stabilizing agent comprises at least one cryopreserving agent.-   79. The method of any one of embodiments 71 to 78, further    comprising combining said genetically modified LAB, said dried    composition, or said freeze-dried composition with a    pharmaceutically acceptable carrier to form a pharmaceutical    composition.-   80. The method of any one of embodiments 76 to 79, further    comprising formulating said dry composition, said freeze-dried    composition or said pharmaceutical composition into a pharmaceutical    dosage form.-   81. A genetically modified bacterium prepared by the method of any    one of embodiments 71 to 80.-   82. A method for preparing a pharmaceutical composition comprising    contacting a culture of the LAB of any one of embodiments 1 to 56    with at least one stabilizing agent forming a bacterial mixture.-   83. The method of embodiment 82, further comprising removing water    from said bacterial mixture, thereby forming a dried composition.-   84. The method of embodiment 82 or 83, comprising freeze-drying said    bacterial mixture thereby forming a freeze-dried composition.-   85. The method of any one of embodiments 82 to 84, wherein said at    least one stabilizing agent comprises at least one cryopreserving    agent.-   86. The method of embodiment 84 or 85, further comprising contacting    said freeze-dried composition with a pharmaceutically acceptable    carrier forming a pharmaceutical composition.-   87. The method of any one of embodiments 84 to 86, further    comprising formulating said freeze-dried composition into a    pharmaceutical dosage form.-   88. The method of embodiment 87, wherein said pharmaceutical dosage    form is selected from the group consisting of a tablet, a capsule, a    granule, and a sachet.-   89. A unit dosage form comprising the LAB of any one of embodiments    1 to 56, the composition of embodiment 57, or the pharmaceutical    composition of embodiment 58.-   90. The unit dosage form of embodiment 89, wherein said unit dosage    form is an oral dosage form.-   91. The unit dosage form of embodiment 90, wherein said oral dosage    form is selected from the group consisting of a tablet, a capsule, a    granule, and a sachet.-   92. The unit dosage form of any one of embodiments 89 to 91, wherein    said unit dosage form comprises from about 1×10⁶ to about 1×10¹²    colony forming units (cfu) of said LAB.-   93. The unit dosage form of embodiment 92 comprising from about    1×10⁸ to about 1×10¹¹ cfu.-   94. A kit comprising (1) an LAB according to any one of embodiments    1 to 56, a composition according to embodiment 57, a pharmaceutical    composition of embodiment 58, or a unit dosage form of any one of    embodiments 89 to 93, and (2) instructions for administering said    LAB, said composition, said pharmaceutical composition, or said unit    dosage form to a mammal.-   95. The kit of embodiment 94, wherein said mammal is a human.-   96. A bacterial suspension comprising the LAB of any one of    embodiments 1 to 56, a solvent, and a stabilizing agent.-   97. The bacterial suspension of embodiment 96, wherein said solvent    is selected from water, oil, and any combination thereof.

1-26. (canceled)
 27. A recombinant lactic acid bacterium (LAB)comprising two chromosomally integrated exogenous nucleic acids, whereinthe first exogenous nucleic acid encodes a human interleukin-10(hIL-10), and wherein the second exogenous nucleic acid encodes a humanproinsulin (hPINS).
 28. The recombinant LAB of claim 27, constitutivelyexpressing and secreting said hIL-10 and said hPINS.
 29. The recombinantLAB of claim 27, wherein the recombinant LAB is a recombinantLactococcus lactis.
 30. The recombinant LAB of claim 27, wherein saidfirst exogenous nucleic acid has at least one feature selected from thegroup of features consisting of: a) comprising a nucleotide sequence atleast 95% identical to SEQ ID NO: 2; b) is transcriptionally regulatedby an hllA promoter (PhllA); and c) is chromosomally integrated at thethyA locus of said recombinant LAB.
 31. The recombinant LAB of claim 27,wherein said hIL-10 has at least one feature selected from the group offeatures consisting of: a) is expressed as a fusion protein comprising aUsp45 secretion leader (SSusp45); b) comprising a proline (Pro) toalanine (Ala) substitution at position 2 of a mature hIL-10; and c)comprising an amino acid sequence at least 95% identical to SEQ IDNO:
 1. 32. The recombinant LAB of claim 27, wherein said secondexogenous nucleic acid has at least one feature selected from the groupof features consisting of: a) comprising a nucleotide sequence at least95% identical to SEQ ID NO: 4; and b) is inserted in a firstpolycistronic expression cassette comprising, in 5′ to 3′ order, a gapBpromoter, a gapB gene, a nucleic acid sequence encoding a Usp45secretion leader (SSusp45), and said second exogenous nucleic acid,wherein the first polycistronic expression cassette expresses a hPINSfusion protein comprising the Usp45 secretion leader (SSusp45).
 33. Therecombinant LAB of claim 32, wherein the first polycistronic expressioncassette further comprises, between said gapB gene and said secondexogenous nucleic acid, an intergenic region that is immediately 5′ to arpmD gene.
 34. The recombinant LAB of claim 27, wherein said hPINS hasat least one feature selected from selected from the group of featuresconsisting of: a) comprising an amino acid sequence at least 95%identical to SEQ ID NO: 3; and b) is expressed as a fusion proteincomprising a Usp45 secretion leader (SSusp45).
 35. The recombinant LABof claim 27, further comprising at least one feature selected from thegroup of features consisting of: a) expressing an exogenous trehalose6-phosphate phosphatase; b) constitutively overexpressing a geneencoding at least one trehalose transporter; c) lacking trehalose6-phosphate phosphorylase (TrePP) activity; and d) lackingcellobiose-specific PTS system IIC component (PtcC) activity.
 36. Therecombinant LAB of claim 35, wherein said trehalose 6-phosphatephosphatase is an OtsB from Escherichia coli; wherein said at least onetrehalose transporter is encoded by aptsI gene and aptsII gene; whereinsaid recombinant LAB comprises an inactivated endogenous trePP, so thatthe recombinant LAB lacks TrePP activity; and wherein said recombinantLAB comprises an inactivated endogenous ptcC, so that the recombinantLAB lacks PtcC activity.
 37. The recombinant LAB of claim 29,comprising: a) an expression cassette chromosomally integrated at thethyA locus of said recombinant LAB, wherein said expression cassettecomprises an hllA promoter (PhllA) that transcribes a nucleic acidsequence encoding a Usp45 secretion leader (SSusp45) and said firstexogenous nucleic acid, and wherein said hIL-10 is expressed as a fusionprotein comprising said SSusp45; b) a first chromosomally integratedpolycistronic expression cassette comprising, in 5′ to 3′ order, a gapBpromoter, a gapB gene, a nucleic acid sequence encoding a Usp45secretion leader, and said second exogenous nucleic acid, wherein saidhPINS is expressed as a fusion protein comprising said SSusp45; c) asecond chromosomally integrated polycistronic expression cassettecomprising, in 5′ to 3′ order, a usp45 promoter, a usp45 gene, anintergenic region that is immediately 5′ to a rpmnD gene, and a thirdexogenous nucleic acid encoding an OtsB from Escherichia coli; d) athird polycistronic expression cassette comprising, in 5′ to 3′ order,an hllA promoter (PhllA) and a nucleic acid comprising an pstI gene andan pstII gene; e) an inactivated endogenous trePP gene, wherein saidtrePP gene is inactivated by gene deletion, so that the recombinant LABlacks TrePP activity; and f) an inactivated endogenous ptcC gene,wherein said ptcC gene is inactivated by insertion of a premature stopcodon, so that the recombinant LAB lacks PtcC activity.
 38. Apharmaceutical composition comprising the recombinant LAB of claim 27and a pharmaceutically acceptable carrier.
 39. A method for thetreatment of type-1 diabetes (T1D) in a human in need thereof comprisingadministering to said human a therapeutically effective amount of: a)the recombinant LAB of claim 27; or b) a pharmaceutical compositioncomprising the recombinant LAB of claim 27 and a pharmaceuticallyacceptable carrier.
 40. The method of claim 39, wherein said human hasrecent-onset T1D.
 41. The method of claim 39, wherein the blood of saidhuman is positive for an auto-antibody selected from the groupconsisting of: an insulin autoantibody (IAA), an islet-cellauto-antibody, a glutamic acid decarboxylase (GAD65) auto-antibody, andan ICA512 antibody.
 42. The method of claim 41, wherein the blood ofsaid human is positive for said IAA.
 43. The method of claim 39, furthercomprising measuring an amount of an insulin autoantibody (IAA) in theblood of said human.
 44. The method of claim 43, wherein the amount ofsaid IAA in the blood of said human is indicative of T1D progression oran outcome of said treatment of T1D.
 45. The method of claim 43, whereinmeasuring the amount of said IAA occurs: a) before administering therecombinant LAB or the pharmaceutical composition to predict an outcomeof said treatment of T1D; or b) after administering the recombinant LABor the pharmaceutical composition to monitor and measure an outcome ofsaid treatment of T1D.
 46. The method of claim 39, further comprisingadministering to said human an immunomodulatory agent.
 47. The method ofclaim 46, wherein said immunomodulatory agent is an anti-CD3 antibody.